Estrogen Replacement Therapy Increases Plasma Ghrelin Levels

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The Journal of Clinical Endocrinology & Metabolism 90(5):2954 –2963 Copyright © 2005 by The Endocrine Society doi: 10.1210/jc.2004-2016

Estrogen Replacement Therapy Increases Plasma Ghrelin Levels Eija Kellokoski, Seppo M. Po¨ykko¨, Anna H. Karjalainen, Olavi Ukkola, Jorma Heikkinen, Y. Antero Kesa¨niemi, and Sohvi Ho¨rkko¨ Department of Internal Medicine (E.K., S.M.P., A.H.K., O.U., Y.A.K., S.H.) and Biocenter Oulu (E.K., S.M.P., O.U., Y.A.K., S.H.), University of Oulu, 90014 Oulu, Finland; and Oulu Deaconess Institute (J.H.), 90100 Oulu, Finland Ghrelin is a novel peptide hormone that has GH releasing activity and also other endocrine and metabolic functions. The purpose of this study was to investigate the effects of estrogen replacement therapy on plasma active ghrelin levels in 64 hysterectomized postmenopausal women receiving peroral estrogen (PE) or transdermal estrogen therapy for 6 months. Active ghrelin was measured using commercial RIA. Estrogen therapy increased plasma active ghrelin from 479 ⴞ 118 to 521 ⴞ 123 pg/ml (P ⴝ 0.002) among all the study subjects. PE therapy increased plasma ghrelin levels from 465 ⴞ 99 to 536 ⴞ 104 pg/ml (P ⴝ 0.001). Transdermal estrogen therapy did not increase plasma ghrelin levels significantly (from 491 ⴞ

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STROGEN THERAPY HAS been available to postmenopausal women for more than 60 yr. Proven benefits include relief of vasomotor symptoms and vaginal atrophy and prevention and treatment of osteoporosis. Early observational studies primarily examining unopposed estrogen preparations suggested that estrogen therapy reduces coronary events (1, 2). Use of estrogen replacement therapy (ERT) has been associated with several antiatherogenic effects such as lower blood pressure (BP) (3), lower plasma total cholesterol levels, and higher high-density lipoprotein (HDL) cholesterol levels (4 – 6). ERT has been shown to improve glucose tolerance (7) and decrease susceptibility of low-density lipoprotein (LDL) to oxidation (8). Furthermore, ERT has been demonstrated to have fibrinolytic activity (4, 9). A recent primary prevention study (The Women’s Health Initiative) showed that use of estrogen increases the risk of stroke but does not affect coronary heart disease incidence (10). A randomized placebo-controlled secondary prevention clinical trial demonstrated no protective effect of hormone replacement therapy (HRT) against cardiovascular disease (11, 12). Nevertheless, estrogen has both rapid and long-term effects First Published Online February 15, 2005 Abbreviations: ANP, Atrial natriuretic peptide; BMI, body mass index; BNP, B-type natriuretic peptide; BP, blood pressure; CV, coefficient of variation; ERT, estrogen replacement therapy; HDL, high-density lipoprotein; HRT, hormone replacement therapy; IGFBP-1, IGF binding protein 1; ISI (composite), whole-body insulin sensitivity index; ISIest, insulin sensitivity index; LDL, low-density lipoprotein; MCRest, metabolic clearance rate of glucose; NT-proANP, N-terminal fragment of proANP; PE, peroral estrogen; TE, transdermal estrogen; VLDL, very low-density lipoprotein. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

132 to 509 ⴞ 138 pg/ml; P ⴝ 0.332). The relative changes in plasma ghrelin levels were associated with the relative changes in serum estradiol concentrations (r ⴝ 0.299; P ⴝ 0.017). During the estrogen therapy, negative associations were found between plasma active ghrelin levels and several plasma lipids (total cholesterol, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, total triglycerides, and very low-density lipoprotein triglycerides). As a conclusion, estrogen replacement therapy increased active plasma ghrelin levels, particularly PE therapy. Additional studies are needed to determine the possible underlying mechanisms. (J Clin Endocrinol Metab 90: 2954 –2963, 2005)

on blood-vessel wall (13–16), but the mechanisms that mediate these effects are not fully understood. Therefore, more studies to provide detailed information on the use of welldefined forms of estrogen are urgently needed. Ghrelin is a novel polypeptide hormone that was identified as an endogenous ligand for an orphan receptor termed GH secretagogue receptor (17). The most abundant source of ghrelin is the stomach (17), but it is expressed at lower levels in various tissues, including small bowel, pancreas, kidney, ovary, pituitary, and hypothalamus (18 –22). Ghrelin was first shown to stimulate the release of GH from the pituitary (17), but it has since been shown to alter feeding behavior, energy metabolism (23, 24), and gastropancreatic functions (25, 26). Ghrelin is down-regulated in human obesity and up-regulated under conditions of negative energy balance such as anorexia nervosa (27). Recent data also suggest that ghrelin may play a significant role in cardiovascular (28), reproductive (29), and endocrine systems (30). Several studies suggest that ghrelin has direct vasodilatory effects (31–33). Animal studies have shown that estrogen is involved in the regulation of ghrelin secretion (34). Estrogen administration decreased the number of ghrelin-producing cells, ghrelin mRNA level in the stomach, and plasma ghrelin levels in ovariectomized rats (34). In addition, ghrelin and estrogen receptor ␣ immunoreactivities were demonstrated in the same cells, suggesting that estrogen may have a direct effect on ghrelin expression (34). Recently the use of oral contraceptives containing estrogen and progesterone was shown to increase plasma total ghrelin levels in severe undernutrition (35). The primary purpose of the present study was to investigate the effects of pure estrogen on the active form of plasma ghrelin in postmeno-

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Kellokoski et al. • Estrogen and Ghrelin

J Clin Endocrinol Metab, May 2005, 90(5):2954 –2963

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pausal women having normal nutritional balance. A secondary purpose was to assess whether ghrelin was associated with parameters of lipid, glucose, and insulin metabolism, BP, or natriuretic peptides.

fasting morning plasma ghrelin concentrations, which have been previously shown to correlate strongly with the 24-h integrated area under the curve values (37).

Subjects and Methods Experimental subjects

Sex hormones, ghrelin, and IGF binding protein-1 (IGFBP-1) were determined at baseline and after 6 months of ERT. Serum FSH and LH were analyzed using an immunofluorometric method (DELFIA; Wallac, Turku, Finland). Serum estrone, estradiol, SHBG, and free testosterone were determined by RIA using the commercial kits (Orion Diagnostica, Espoo, Finland). Intra- and interassay CV for estradiol in our analyses were 2.8 and 5.8% at baseline and 3.5 and 8.1% after 6 months of therapy, respectively. Plasma IGFBP-1 was measured with an immunoenzymometric assay (Medix Biochemica, Kauniainen, Finland) as described previously (38). Plasma atrial natriuretic peptide (ANP), N-terminal fragment of proANP (NT-proANP), and B-type natriuretic peptide (BNP) concentrations were determined with specific RIAs as previously described (3). Plasma aldosterone was also analyzed by RIA (Diagnostic Products Corp., Los Angeles, CA) and renin by immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA).

A total of 105 postmenopausal women seeking hormone substitution therapy for climacteric symptoms were originally screened. Seventynine met the inclusion criteria and volunteered to participate in the study. There were four drop-outs in the PE group and two in the TE group during the study. Thirty-nine women received peroral estrogen (PE) and 40 received transdermal estrogen (TE). Plasma samples of 64 participants were available (29 for PE and 35 for TE) for our ghrelin measurement. These subjects had been randomized to receive either peroral estradiol or transdermal estradiol gel for 6 months as described earlier in detail (6, 36). Briefly, because the study was focused on the effects of estrogen alone, hysterectomized women were chosen to avoid the need of progesterone for endometrium protection. The study was conducted using a double-blind and double-dummy design. The PE group (n ⫽ 29) received a daily tablet containing 2 mg estradiol valerate (Orion Pharma, Espoo, Finland) and placebo gel. The TE group (n ⫽ 35) received estradiol gel (Divigel, Orion Pharma; Sandrena, NV Organon, Oss, The Netherlands), which was packed as daily dose units of 1.0 g in stick-pack sachets containing 1.0 mg 17␤-estradiol (0.1% gel) and placebo tablets. The criteria for inclusion were as follows: 45– 65 yr of age, a previous hysterectomy with at least one remaining ovary, serum FSH more than 30 IU/liter, fasting blood glucose less than 6.7 mmol/liter, BP less than 160/95 mm Hg and body mass index (BMI) less than 30 kg/m2. The time from menopause was evaluated based on the information on when menorrhea was known to end. This information was obtained from the study participants. If this was not applicable (i.e. if the subjects had undergone hysterectomy before menopause), the time from menopause was based on symptoms. Women having contraindications to estrogen therapy or any diseases or medication interfering with lipid metabolism were excluded. The participants were able to keep their lifestyle and concomitant medication during the study. Height, weight, and waist and hip circumferences were measured. BMI and waist-to-hip ratio (waist circumference in centimeters/hip circumference in centimeters) were used to estimate generalized and abdominal obesity, respectively. BP measurements were made by using an automatic, microprocessor-controlled device (Critikon Dinamap 1846 SX/P; Critokon Inc., Tampa, FL) (3). Written informed consent was obtained from all subjects. The study was approved by the Ethical Committee of the Faculty of Medicine, University of Oulu (Oulu, Finland), and followed the Declaration of Helsinki.

Blood sampling

Hormone assays

Blood glucose and insulin Blood samples were obtained after an overnight fast for a standard 75-g oral glucose tolerance test. In the oral glucose tolerance test, fasting and postchallenge blood glucose, serum insulin, and C-peptide were determined at 30, 60, and 120 min. Blood glucose was measured with the glucose dehydrogenase method (Merck no. 12194; Merck, Darmstadt, Germany). Serum C-peptide concentrations were determined using a commercial double-antibody RIA (Double Antibody C-peptide; EURO/ DPC Ltd., Llanberis, UK), and serum insulin levels were measured with two-site immunoenzymometric assay (AIA-PACK IRI; Tosoh Corp., Tokyo, Japan). GhbA1c was determined by liquid chromatography. Calculations for whole-body insulin sensitivity index [ISI (composite)], insulin sensitivity index (ISIest) and metabolic clearance rate of glucose (MCRest) have been previously described (39).

Lipids and lipoproteins Determination of plasma lipids and lipoproteins and LDL clearance studies were carried out as previously described in detail (36). Total plasma cholesterol and triglyceride levels were determined by enzymatic colorimetric methods (kits from Boehringer Diagnostica, Mannheim, Germany). Very low-density lipoprotein (VLDL), HDL, and LDL were isolated by repeated ultracentrifugations according to density. HDL cholesterol was determined from VLDL-free plasma after precipitations of LDL with heparin-manganese. LDL cholesterol was also calculated by the Friedewald formula (40), and these values were used in the response analyses.

Blood samples were drawn into EDTA-containing tubes for plasma and serum samples in the morning after an overnight fast at baseline and after 6 months of ERT. Plasma and serum was separated by centrifugation at 1200 ⫻ g (2600 rpm) for 15 min (4 C). After centrifugation, plasma and serum samples were stored at ⫺20 C. Sex hormones and parameters of lipid and glucose metabolism were analyzed after collecting the samples. All the samples were analyzed in one assay for a particular analyte.

Absorption of dietary cholesterol and diet

Measurement of ghrelin

Statistical analysis

Plasma ghrelin levels were measured by commercial RIAs (Linco Research, Inc., St. Louis, MO), using 125I-labeled ghrelin as a tracer and a ghrelin antiserum raised against Ser(3) octanoylated ghrelin for measurement of active form of ghrelin (100% specificity for ghrelin and ghrelin 1–10; ⬍0.1% specificity for ghrelin 14 –28 and des-octanoyl ghrelin). The detection limit for the assay was 10 pg/ml. Intra- and interassay coefficients of variation (CV) reported by the manufacturer were 6.5–9.5 and 9.6 –16.2%, respectively (for conversion to SI units: picograms per milliliter ⫻ 3.371 ⫽ picomoles per liter). Intra- and interassay CV in our analyses were 2.3 and 8.7%, respectively. Even though the secretion of ghrelin is highly variable throughout the day, we used

Data analyses were performed with the software package SPSS for Windows (12.0) (SSPS Inc., Chicago, IL). The results for continuous variables are presented as mean ⫾ sd, unless stated otherwise. To compare the ghrelin responses between the treatment groups, repeated measurements ANOVA was used. The changes from baseline to 6 months were analyzed by paired-sample t test, and the differences between the therapies were compared by independent-sample t test. Log-transformed values of total and VLDL triglycerides were used to normalize the skewed distributions. The correlation between the normally distributed variables was assessed by Pearson correlation coefficient. Spearman rank correlation coefficient was used when the criteria for normal distribution were not achieved. Linear

Measurements and calculations for the absorption of dietary cholesterol are described previously in detail (36). Absorption was measured by the peroral double-isotope continuous-feeding method. Seven-day food records were analyzed by a dietitian with the Finnish Food Database Program, Nutrica (The Social Insurance Institution of Finland, Turku, Finland).

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regression was used to test determinants of the changes in ghrelin concentrations and in plasma lipids. P ⬍ 0.05 (two-sided) was regarded as statistically significant.

Results

In the present study, we measured the active ghrelin levels from 64 study subjects that were a part of a larger study (n ⫽ 79) that has been well documented earlier (3, 6, 36, 38, 39, 41). The main characteristics of the 64 study subjects are presented in Table 1. The study groups did not differ statistically in age, BMI, or menopausal status. The body weight and waist-to-hip ratio remained notably constant after the 6-month treatment on the peroral and the transdermal therapies. Ghrelin and estrogen

ERT significantly increased (P ⫽ 0.002) plasma ghrelin concentrations from 479 ⫾ 118 (mean ⫾ sd) to 521 ⫾ 123 pg/ml among all the study subjects (n ⫽ 64). The increase in plasma ghrelin level was from 465 ⫾ 99 to 536 ⫾ 104 pg/ml (P ⫽ 0.001) among the subjects receiving peroral ERT. No significant increase (from 491 ⫾ 132 to 509 ⫾ 138 pg/ml; P ⫽ 0.332) was seen among the subjects receiving TE (Fig. 1). Even though the increase in ghrelin concentrations was higher in the PE group, the difference in the response between the groups was not statistically significant (P ⫽ 0.061; repeatedmeasures ANOVA). ERT significantly increased serum estrone levels from 204 ⫾ 126 to 1228 ⫾ 1208 pmol/liter (P ⫽ 0.004) among all the subjects. PE therapy increased serum estrone concentration (from 183 ⫾ 98 to 2212 ⫾ 1186 pmol/ liter; P ⬍ 0.001), which was statically more (P ⬍ 0.001; between the therapies) than in the TE therapy (from 221 ⫾ 144 to 412 ⫾ 190 pmol/liter; P ⬍ 0.001; Fig. 1). ERT increased also serum estradiol levels (from 105 ⫾ 165 to 298 ⫾ 182 pmol/ liter, P ⬍ 0.001) among all the study subjects. PE therapy increased serum estradiol levels from 95 ⫾ 145 to 345 ⫾ 194


pmol/liter (P ⬍ 0.001) and from 114 ⫾ 181 to 259 ⫾ 164 pmol/liter (P ⬍ 0.001) with the TE therapy (P ⫽ 0.065 between the groups; Fig. 1). Plasma ghrelin concentrations were not associated with the serum estradiol or estrone levels at baseline or on the therapy. However, the relative changes in plasma ghrelin levels (ERT compared with baseline) were positively related to the relative changes in serum estradiol concentrations among all the study subjects (n ⫽ 64, r ⫽ 0.299, P ⫽ 0.017; Fig. 2). The results were similar when we calculated the absolute changes. We also used a multivariate linear regression analysis. The changes in serum estrone, estradiol, FSH, LH, and testosterone were subjected to a stepwise analysis using the change in plasma ghrelin as the dependent variable. The absolute change in serum FSH and free testosterone remained significantly associated with the change in plasma ghrelin (P ⫽ 0.015 and P ⫽ 0.034, respectively; r2 ⫽ 0.165). However, the relative changes were also analyzed in the same way, but no statistically significant correlations were found. Ghrelin and other hormones

Table 1 shows the values for serum sex hormones, renin, and aldosterone at baseline and on therapy. ERT decreased serum FSH levels (from 63 ⫾ 21 to 35 ⫾ 16 IU/liter; P ⬍ 0.001) among all the study subjects. PE therapy decreased serum FSH levels from 65 ⫾ 21 to 32 ⫾ 14 IU/liter (P ⬍ 0.001) and TE therapy from 62 ⫾ 20 to 37 ⫾ 17 IU/liter (P ⬍ 0.001). Plasma ghrelin levels were not related to serum FSH or free testosterone levels. The level of serum SHBG had a positive association with ghrelin concentration among the subjects receiving TE. This significant correlation was seen at baseline (r ⫽ 0.457, P ⫽ 0.006) and also on therapy (r ⫽ 0.385, P ⫽ 0.022). No associations were found between plasma ghrelin concentrations and serum renin levels or between ghrelin concentrations and serum aldosterone levels.

TABLE 1. Clinical characteristics Peroral estradiol (n ⫽ 29)

Age (yr) Weight (kg) Waist-hip-ratio BMI (kg/m2) Time from menopause (yr) S-estrone (pmol/liter) S-estradiol (pmol/liter) FSH (IU/liter) LH (IU/liter) SHBG (nmol/liter) S-free testosterone (pmol/liter) Ghrelin (pg/ml) Renin (mU/liter) Aldosterone (pmol/liter) Total cholesterol (mmol/liter) LDL cholesterol (mmol/liter) HDL cholesterol (mmol/liter) Total triglycerides (mmol/liter) Systolic BP (mm Hg) Diastolic BP (mm Hg)

Transdermal estradiol (n ⫽ 35)

Baseline

On therapy

Baseline

On therapy

54.0 (⫾3.2) 69.9 (⫾6.4) 0.82 (⫾0.06) 26.8 (⫾2.7) 5.1 (⫾2.9) 183 (⫾98) 95 (⫾145) 65.2 (⫾21) 34.9 (⫾12) 45.9 (⫾24) 3.6 (⫾1.6) 465 (⫾99) 32.9 (⫾36) 316 (⫾149) 6.4 (⫾0.90) 4.3 (⫾0.80) 1.60 (⫾0.37) 1.30 (⫾0.53) 143 (⫾13) 87 (⫾8)

54.6 (⫾3.2) 69.6 (⫾5.7) 0.81 (⫾0.05) 26.8 (⫾2.4) 5.7 (⫾2.9) 2211 (⫾1186)a 345 (⫾194)a 32.3 (⫾15)a 20.6 (⫾9)a 92.7 (⫾51)a 2.4 (⫾0.8)b 536 (⫾104)b 23.8 (⫾23) 403 (⫾203) 5.9 (⫾0.78)a 3.5 (⫾0.79)a 1.80 (⫾0.38)a 1.44 (⫾0.55)b 139 (⫾14) 83 (⫾8)b

54.8 (⫾2.9) 69.1 (⫾7.7) 0.80 (⫾0.05) 26.0 (⫾2.4) 5.8 (⫾3.4) 221 (⫾144) 114 (⫾181) 62.0 (⫾20) 30.7 (⫾11) 44.0 (⫾16) 3.4 (⫾1.6) 491 (⫾132) 27.3 (⫾15) 396 (⫾282) 6.4 (⫾0.87) 4.2 (⫾0.83) 1.60 (⫾0.39) 1.41 (⫾0.59) 138 (⫾22) 83 (⫾9)

55.2 (⫾2.9) 69.7 (⫾8.2) 0.80 (⫾0.05) 26.2 (⫾2.5) 6.4 (⫾3.4) 412 (⫾190)a 259 (⫾164)a 37.2 (⫾17)a 19.5 (⫾9)a 51.1 (⫾26)c 2.8 (⫾1.2) 509 (⫾138) 33.0 (⫾39) 415 (⫾298) 6.0 (⫾0.75)a 3.8 (⫾0.68)a 1.56 (⫾0.35) 1.38 (⫾0.59) 131 (⫾20)b 79 (⫾8)a

Values are mean (⫾SD). a P ⬍ 0.001; b P ⬍ 0.01; and c P ⬍ 0.05 vs. corresponding baseline value.



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FIG. 1. The effect of ERT on plasma active ghrelin (A and B), serum estrone (C and D), and serum estradiol levels (E and F) at baseline and during peroral and TE therapy. The symbols represent mean values (⫾SEM). P values were obtained by paired sample t test.

Ghrelin and measurements of body mass

It has been previously shown that ghrelin levels are lower in obese humans compared with lean (27), and numerous studies have demonstrated a negative correlation between BMI and ghrelin levels (42– 45). Our study also confirms the inverse association between BMI and ghrelin levels on postmenopausal women with and without ERT. Figure 3 shows this negative association between plasma ghrelin levels and BMI at baseline (r ⫽ ⫺0.353, P ⫽ 0.004), on TE therapy (r ⫽ ⫺0.419, P ⫽ 0.012), and on PE therapy (r ⫽ ⫺0.368, P ⫽ 0.050). Ghrelin and lipids

Table 1 shows the different parameters of plasma lipids at baseline and on therapy. PE therapy decreased total cholesterol level from 6.43 ⫾ 0.90 to 5.93 ⫾ 0.78 mmol/liter (P ⬍ 0.001) and transdermal therapy decreased total cholesterol from 6.41 ⫾ 0.87 to 6.02 ⫾ 0.75 mmol/liter (P ⬍ 0.001). Similarly, LDL and VLDL cholesterol concentrations decreased during the ERT (data not shown). Only PE therapy increased plasma triglycerides from 1.30 ⫾ 0.53 to 1.44 ⫾ 0.55 mmol/liter (P ⫽ 0.019). TE therapy did not change triglyceride levels (from 1.41 ⫾ 0.59

to 1.38 ⫾ 0.59; P ⫽ 0.573). Figure 4 shows that during estrogen therapy, plasma ghrelin concentrations were negatively associated with plasma total cholesterol levels (r ⫽ ⫺0.265, P ⫽ 0.034), LDL cholesterol levels (r ⫽ ⫺0.248, P ⫽ 0.048) and plasma total triglyceride levels (r ⫽ ⫺0.307, P ⫽ 0.013) among all the study subjects. At the baseline, the association was statistically significant only between plasma ghrelin levels and plasma total triglycerides (r ⫽ ⫺0.345, P ⫽ 0.005; Fig. 4). Significant negative correlations were also seen between plasma ghrelin levels and VLDL cholesterol levels (r ⫽ ⫺0.303, P ⫽ 0.007; Fig. 5) and between plasma ghrelin levels and VLDL triglyceride levels (r ⫽ ⫺0.372, P ⫽ 0.002; Fig. 5) at baseline and during estrogen therapy among all the subjects (Fig. 5). HDL cholesterol levels increased from 1.60 ⫾ 0.37 to 1.81 ⫾ 0.38 mmol/liter (P ⬍ 0.001) on PE therapy. No changes in HDL levels were seen on TE therapy. HDL cholesterol levels showed no association with plasma ghrelin levels among our study subjects. We also used a multivariate regression analysis to measure the descriptive factors for metabolic parameters. No independent variable correlated with the absolute or relative

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change LDL clearance (from 0.306 ⫾ 0.045 to 0.310 ⫾ 0.042 pools per day; P ⫽ 0.508). Ghrelin levels showed no associations with LDL clearance rates. ApoB was associated with ghrelin levels at baseline (r ⫽ ⫺0.262, P ⫽ 0.036) and during therapy (r ⫽ ⫺0.334, P ⫽ 0.007) (data not shown). Glucose and insulin metabolism

FIG. 2. Correlation between relative changes (ERT values compared with baseline values, in percentages) in serum estradiol levels and in plasma active ghrelin levels during estrogen replacements therapy. The open (E) and filled (F) circles represent subjects receiving PE and TE therapy, respectively.

changes in plasma total cholesterol or triglycerides. The absolute changes in serum estradiol (P ⫽ 0.01), estrone (P ⫽ 0.026), and free testosterone (P ⫽ 0.01) were the variables that correlated significantly with the absolute changes in plasma HDL among all the subjects. This was not seen when the therapies were analyzed separately. Also, the absolute changes in plasma LDL cholesterol associated with the absolute changes in serum estrone. The absolute changes in both VLDL cholesterol and VLDL triglycerides correlated significantly with the absolute changes in serum FSH (P ⫽ 0.029 for both). Absorption of dietary cholesterol

Absolute absorption of dietary cholesterol decreased from 1.96 ⫾ 1.09 to 1.53 ⫾ 0.96 mg/kg䡠d (P ⫽ 0.019) during PE therapy and from 2.03 ⫾ 0.86 to 1.71 ⫾ 0.72 mg/kg䡠d (P ⫽ 0.035) during TE therapy. Ghrelin levels did not associate with the absolute absorption of dietary cholesterol. No correlation was observed between the changes in plasma ghrelin levels and cholesterol absorption. LDL clearance rate

PE therapy increased LDL clearance rates from 0.295 ⫾ 0.037 to 0.343 ⫾ 0.044 pools per day (P ⬍ 0.001). TE did not

FIG. 3. Correlations between plasma active ghrelin levels and BMI at baseline (A) and after 6 months transdermal (B) and after 6 months PE therapy (C). The open (E) and filled (F) circles represent subjects receiving PE and TE therapy, respectively.

Fasting insulin concentrations and insulin resistance have been associated with ghrelin levels in several studies (42, 46 – 48). Our findings are partly in accordance with these previous results, and Table 2 lists the associations between plasma ghrelin levels and insulin and glucose metabolism parameters at baseline and on PE and TE therapy. At baseline, none of the parameters associated with plasma concentrations of active ghrelin among the subjects receiving PE. However, plasma active ghrelin associated significantly with fasting insulin, glucose and C-peptide, GHbA1c (glycosylated hemoglobin), ISI (composite), ISIest, and MCRest among subjects receiving TE therapy (Table 2). After 6 months of therapy, plasma ghrelin was significantly associated with fasting glucose and ISI (composite) on PE therapy (Table 2). Only fasting C-peptide remained significantly correlated with plasma active ghrelin during TE therapy (Table 2). In addition, IGFBP-1 levels associated with plasma active ghrelin during the TE therapy (Table 2). BP, ANP, and BNP

Both PE and TE therapy reduced systolic and diastolic BP. Systolic BP decreased from 143 (⫾14) to 139 (⫾14) mm Hg (P ⫽ 0.066) on PE therapy and from 138 (⫾23) to 131 (⫾20) mm Hg (P ⫽ 0.001) on TE therapy. Diastolic BP decreased from 87 (⫾8) to 83 (⫾8) mm Hg (P ⫽ 0.004) in PE therapy and from 83 (⫾9) to 79 (⫾8) mm Hg (P ⬍ 0.001) in TE therapy. Plasma active ghrelin levels or relative changes in plasma active ghrelin levels were not statistically associated with systolic and diastolic BP or changes in the BP, although some trend was found between the active ghrelin levels and diastolic BP. Also, ANP, BNP, and NT-proANP levels were not related to active ghrelin levels (data not shown). Diet

The subjects followed their habitual diets throughout the study (data obtained from food records). Associations between active ghrelin levels and different nutrient levels were not seen among these study subjects (data not shown).



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FIG. 4. Correlations between plasma active ghrelin levels and total cholesterol (A and B), LDL cholesterol (C and D) and total triglycerides (E and F) at baseline and during estrogen therapy. The open (E) and filled (F) circles represent subjects receiving PE and TE therapy, respectively.

Discussion

The major finding of the present study was that postmenopausal ERT increases plasma active ghrelin levels. In addition, when our study subjects were placed on the ERT, the relative changes in their plasma levels of active ghrelin were positively associated with the relative changes in serum estradiol concentrations. Our study investigates the effects of estrogen replacement on plasma active ghrelin levels in postmenopausal women. The ERT regimens, not in combination with progesterone, allowed us to study estrogen alone. PE therapy increased

FIG. 5. Correlations between plasma active ghrelin levels and VLDL cholesterol (A and B) and VLDL triglycerides (C and D) at baseline and on estrogen therapy. The open (E) and filled (F) circles represent subjects receiving PE and TE therapy, respectively.

plasma active ghrelin levels, but TE therapy did not change ghrelin levels significantly. Serum estradiol concentrations were quite similar between the study groups and both therapies significantly increased serum estradiol concentrations. To our surprise, even though the increases in plasma ghrelin concentrations were higher in the PE group, the difference in the ghrelin responses between these two different groups (PE vs. TE) was not statistically significant (P ⫽ 0.061; repeatedmeasures ANOVA). The relative changes in estradiol levels correlated with the relative changes in active ghrelin levels (and the results were similar when we calculated the absolute

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TABLE 2. Relationships (r values) between plasma ghrelin levels and measures of glucose and insulin metabolism at baseline and on peroral and transdermal therapy Plasma ghrelin Peroral estradiol (n ⫽ 29)

Fasting glucose Fasting insulin Fasting C-peptide GHbA1c ISI (composite) ISIest MCRest IGFBP-1

Transdermal estradiol (n ⫽ 35)

Baseline

On therapy

Baseline

On therapy

0.024 – 0.035 0.118 0.175 0.003 – 0.216 0.093 – 0.111

– 0.393a – 0.338 – 0.328 0.212 0.513a 0.092 0.149 0.104

– 0.336a – 0.379a – 0.356a – 0.410a 0.332a 0.346a 0.343a 0.233

– 0.274 – 0.219 – 0.355a 0.066 0.287 0.319 0.191 0.389a

GHbA1c, Glycosylated hemoglobin. a P ⬎ 0.05. P values obtained by Pearson correlation coefficient.

changes). In contrast, the increase in serum estrone was markedly higher in PE therapy compared with TE therapy, and serum estrone levels or the changes in serum estrone levels were not related to the ghrelin levels. Because in the present study we have not examined the possible underlying mechanisms for our findings, we can only speculate that the differences in serum estrone levels might be a causative factor for the different increases in plasma ghrelin levels between PE and TE therapy. According to our multivariate analysis, serum FSH and free testosterone might also have an effect on the changes in plasma active ghrelin. In contrast, the route of administration can also have a role in the results. Because ghrelin is produced by the stomach, PE might have a direct effect on the ghrelin-producing cells in the stomach. Animal studies have shown previously that estrogen is involved in the regulation of ghrelin secretion (34). Estrogen administration of ovariectomized rats decreased the number of ghrelin-producing cells, decreased ghrelin mRNA levels in the stomach, and decreased plasma total ghrelin levels (34). In addition, the same study demonstrated that ghrelin and estrogen receptor ␣ were colocalized in the stomach, suggesting that estrogen may have a direct effect on ghrelin expression (34). Our results diverged with these findings. It may be explained partly by the differences in species; also the length of the animal study was shorter (3 d) and the estrogen was administrated to ovariectomized rats by implanted capsules filled with 17␤-estradiol benzoate (34). Several human studies on the interaction between ghrelin and sex hormones are already available. High androgen levels have been shown to associate with low total ghrelin levels in women with polycystic ovary disease (49). Furthermore, when these women were treated with antiandrogen therapy, their plasma total ghrelin levels increased significantly (50). Also, testosterone therapy has been shown to increase plasma total ghrelin levels in men with hypogonadism (51). An earlier cross-sectional study did not demonstrate differences in plasma total ghrelin levels between postmenopausal women with and without HRT (52). The differences between this study (52) and our study may be explained by different ages, status of menopause, or the type of HRT. Also, in the previous study (52), only 11 postmenopausal women were on HRT and the cross-sectional study did not establish a causative role for HRT. Our study design allowed us to study

the effects of estrogen itself on ghrelin levels. One additional explanation for this and other contradictory findings may be that we have measured plasma active ghrelin, whereas most of the other reports measured total ghrelin. Very recently, the use of oral contraceptives containing both estrogen and progesterone was shown to increase plasma total ghrelin levels in severe undernutrition (35). The authors suggested that these changes might be due to effects of estrogen (35). In the present study, we have confirmed these positive effects of estrogen alone on plasma active ghrelin levels during normal nutritional balance. However, the mechanisms underlying these findings remain unknown. In the present study, we analyzed circulating levels of plasma octanoylated ghrelin using antibody that recognizes an octanoylated ghrelin. An acylated form of ghrelin has been found to be an active form of ghrelin that has endocrine activity (17, 53). Nonacylated ghrelin is unable to bind to a type 1a GH secretagogue receptor (54) and is not characterized by endocrine activities (55), although nonendocrine functions have been reported for the nonacylated form of ghrelin (56). A recent study investigated whether nonacylated ghrelin is likely to counterbalance the influence of acylated ghrelin. However, an acylated ghrelin and total ghrelin are well correlated (57), and the ratio of these two remains constant under a wide variety of conditions (58). Therefore, we found that the measurement of only octanoylated ghrelin is sufficient for our purposes. Many animal studies have been discovered the effects of estradiol on eating by controlling meal size (59 – 61). Estradiol treatment has been demonstrated to decrease daily food intake, adiposity, and body weight, and the behavioral and neural responsivity of the cholecystokinin satiation-signaling system in ovariectomized mice (62). Similarly, ghrelin has a role in signaling hunger and meal initiation. The detailed mechanism of how estradiol acts in satiation signaling is not fully understood. These previous studies suggest that we cannot exclude the possibility that in our study estradiol modulated the eating behavior of our study subjects through ghrelin signaling. However, the body weights of our study subjects did not change significantly during the study and the plasma ghrelin levels were not associated with the diets of the study subject (evaluated by the food records), suggesting that this kind of modulation did not occur in our study. An immobilized form of ghrelin has been shown to bind specifically to a species of HDL (63). In population studies, ghrelin concentrations have been found to associate positively to HDL cholesterol levels and LDL particle sizes (64). However, we found no associations between HDL and ghrelin levels among our study subjects. A recent novel investigation demonstrated that administration of ghrelin into rats increased the liver triglyceride content and altered the expression of several genes involved in lipogenesis and glucogenesis (genes such as acetyl-coenzyme A carboxylase, fatty acid synthase, carnitine palmitoyl transferase-I, mitochondrial uncoupling proteins, and peroxisome proliferatoractivated receptor ␥) (65). These new interesting findings strongly suggest that ghrelin may have an important role in lipid metabolism. Indeed, we found negative associations between plasma ghrelin levels and total cholesterol, LDL


cholesterol, VLDL cholesterol, total triglyceride, and VLDL triglyceride levels. Because estrogen therapy itself has an effect on these plasma lipid levels, the effect of ghrelin cannot be evaluated separately. We would like to emphasize that our observations are merely associations between plasma active ghrelin and parameters of plasma lipids, and because we have not examined the possible underlying mechanisms for these findings, we can only speculate on these associations. Ghrelin has been localized to the medial layer of human blood vessels (32). Bolus injections of ghrelin have been shown to cause a significant decrease in the mean arterial pressure in healthy volunteers (31). It has also been demonstrated that ghrelin is an effective endothelium-independent vasodilator of human arteries (66). Also, ghrelin administration has been shown to attenuate hypertension and right ventricular hypertrophy in rats (67). In contrast, a recent study demonstrated that ghrelin infusion dose-dependently increased coronary perfusion pressure, constricted coronary arterioles, and significantly enhanced the pressure-induced myogenic tone of arterioles in rats (68). In this study, ghrelin had no effect on the secretion or on the gene expression of ANP or BNP (68), which are BP-related peptides having natriuretic, diuretic, and vasodilatory activity (69). We and others previously demonstrated an inverse relationship between plasma ghrelin levels and BP (46, 64). In the present study, however, we found no association between ghrelin levels and BP among the postmenopausal women, a population that consisted of normotensive women (n ⫽ 64). The range of BP in our present study is quite narrow when compared with our previous large (n ⫽ 1024) population, which consisted of both normotensive controls and hypertensive subjects. The narrower range of BP and the smaller number of subjects may be reasons explaining why the statistical significance could not be found between ghrelin level and BP in this study. However, some in significant trend was found in the correlation. In addition, no relation was found between the ghrelin levels and plasma ANP, BNP, or NT-proANP levels. Regulation of GH secretion includes a dual system with GHRH and somatostatin. GHRH stimulates while somatostatin inhibits GH secretion. In addition, a third independent pathway involved in the regulation of GH secretion consists of ghrelin. Several other hormones have been shown to affect GH secretion. Among these, estrogen has been shown to increase GH secretion through mechanisms that are not currently well known. Estrogen treatment has been shown to increase serum GH concentrations in postmenopausal (70) and prepubertal hypogonadal female subjects (71) and to enhance the pulsatile drive of GH secretion. It has been suggested that ghrelin may mediate the well-known effects of estrogen on the GH axis (72). A recent study by Broglio et al. (30) demonstrated in rats that the GH-releasing effect of ghrelin decreases with age. In humans it has not yet been decisively established what happens to plasma ghrelin levels with age. In Prader-Willi syndrome, ghrelin levels have been shown to be negatively correlated with age (73), yet another study (52) found a statistically significant positive relation between age and plasma ghrelin levels. In the present study, we examined the


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effect of estrogen therapy on plasma active ghrelin levels in a prospective study design that lasted for 6 months and we did not expect the aging to affect the plasma ghrelin levels during the study. In summary, we have demonstrated that ERT, particularly when administered perorally, increases plasma ghrelin levels among the postmenopausal female subjects. The clinical implications of increased ghrelin in response to ERT are not currently known. Additional studies are needed to investigate the possible underlying mechanisms. Acknowledgments We are grateful to Prof. Heikki Ruskoaho (M.D., Ph.D.) and Prof. Olli Vuolteenaho (M.D., Ph.D.) for the measurement of ANP, BNP, and NT-proANP levels, and Marita Paassilta (M.D., Ph.D.) for analysis of plasma IGF-1 and IGFBP 1 concentrations. Received October 13, 2004. Accepted February 3, 2005. Address all correspondence and requests for reprints to: Eija Kellokoski, Clinical Research Center, Department of Internal Medicine, University of Oulu, P.O. Box 5000, FIN-90014 Oulu, Finland. E-mail: [email protected]. This work was supported by The Research Council for Health of the Academy of Finland and the Finnish Foundation for Cardiovascular Research.

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