Assessment of ghrelin and leptin receptor levels in postmenopausal women who received oral or transdermal menopausal hormonal therapy

Abstract

Objective: In postmenopausal women, an increased leptin concentration and reduced levels of ghrelin and adiponectin were observed. The aim of this study was to evaluate the concentrations of the active form of ghrelin, total ghrelin, leptin receptor, lipoprotein(a) (Lp(a)), and plasminogen activator inhibitor type 1 (PAI-1) in postmenopausal women who received oral or transdermal menopausal hormonal therapy (MHT). Methods: The study involved 76 healthy women: 46 women aged from 44 to 58 years who received oral (26) or transdermal (20) MHT; the control group consisted of 30 women aged from 44 to 54 years who did not receive MHT. The plasma concentrations of total ghrelin, the active form of ghrelin, Lp(a), and PAI-1:Ag were measured by enzyme-linked immunosorbent assay (ELISA). The concentration of the leptin receptor was measured by enzyme immunometric assay (EIA). Results: We observed a significantly higher concentration of total ghrelin and the active form of ghrelin in women who received transdermal MHT in comparison with those who took oral MHT. We also found a significantly lower concentration of total ghrelin in women who received oral MHT compared with the control group. A higher concentration of PAI-1:Ag was found in the group of women who took transdermal MHT in comparison with those who took oral MHT and with the control group. The differences were statistically significant. Additionally, we found a significant negative correlation between the concentrations of total ghrelin and PAI-1:Ag and a positive correlation between the concentrations of total ghrelin and leptin receptor in women who received transdermal MHT. Conclusions: The study showed that women who used transdermal MHT had higher levels of total ghrelin than women who took oral MHT. This indicates a beneficial effect of the transdermal route of MHT. However, transdermal therapy was associated with adverse effects with regard to the observed higher levels of PAI-1:Ag, which in turn, can lead to a reduction in fibrinolytic activity.

1. Introduction

Leptin and ghrelin are antagonistic hormones that regulate and control body weight. Ghrelin is a peptide hormone with a chain of about 28 amino acids. An octanoyl group is attached to the third position of the polypeptide chain, where serine is located. In serum there is also a ghrelin molecule without an octanoyl group, which presumably has no endocrine action. In human gastric mucosa, four ghrelin derivatives have been isolated. They were classified according to the structure of the acyl group at the serine molecule. Peptides which were distinguished either had no substituent (most numerous), or their substituent was an octanoyl (C8:0, active form), decanyl (C10:0) or decenyl (C10:1) group. Ghrelin is synthesized mainly in the stomach but also in small amounts in the placenta, pancreas, kidney, pituitary, and hypothalamus. Its production is also considered as a sign of starvation before having a meal (Meier and Gressner, 2004). Biological activity of ghrelin is possible through two types of receptors, G-protein-coupled receptors types 1 and 1b (growth hormone secretagogue receptor (GHS-R)), which are located in the pituitary, hypothalamus, kidney or adipose tissue (Konturek et al., 2004).

Ghrelin significantly affects the functioning of the digestive tract. Through the vagus nerve it stimulates the motility and secretion of hydrochloric acid by the stomach and inhibits the secretion of pepsin. It plays an important role in the regulation of appetite, energy balance, and glucose homeostasis. The serum ghrelin level increases during starvation, weight loss, and with an increase in blood insulin levels, while it decreases in a period of satiety (Toshinai et al., 2001; Nishi et al. 2005). Furthermore, a circadian rhythm of ghrelin secretion has been observed. Its level increases during fasting, before meals and at night, but decreases after meals, especially in those who are rich in fats and carbohydrates (Weigle et al., 2003).

Leptin is synthesized primarily by adipocytes of white fatty tissue under the skin, including the muscles, placenta, and stomach. Women have higher leptin levels than men due to their higher content of subcutaneous tissue. Biological action of leptin is possible via membrane receptors that belong to the class I cytokine family. The receptors are located in the hypothalamus (where there are appetite control centers), as well as in the thyroid, adrenal glands, and ovaries (Meier and Gressner, 2004; Szumiło et al., 2007). Several isoforms of leptin receptors (Ob-Ra, Ob-Rb, Ob-Rc, Ob-Rd, and Ob-Re) have been identified. It is believed that the Ob-Ra form is the conveyor of leptin, and Ob-Re is a soluble form of the leptin receptor transmembrane. The Ob-Rb form is the only receptor isoform that contains an active intracellular domain (Meier and Gressner, 2004).

Leptin activates processes aimed at reducing energy stores by reducing the demand for food. It affects the body’s energy balance, the maturation of the reproductive system in women, and the process of angiogenesis in men (Meier and Gressner, 2004; Nishi et al., 2005; Szumiło et al., 2007). This protein stimulates the proliferation of colonocytes and belongs to a group of factors that regulate intestinal absorption (Nishi et al., 2005). Leptin is known as a satiety hormone because it suppresses appetite (Szumiło et al., 2007). In most obese people, resistance to leptin has been observed, which may affect the disorder in energy demand (Toshinai et al., 2001; Meier and Gressner, 2004).

After menopause, the incidence of polymetabolic syndrome increases due to the reorganization of adipose tissue and its appearance in increased amounts in the abdominal area. Intra-adipose tissue adipocytes synthesize factors that influence the inflammatory process and increase insulin resistance. These factors include leptin, ghrelin, adiponectin, and plasminogen activator inhibitor type 1 (PAI-1). In postmenopausal women, leptin and PAI-1 concentrations increase whereas ghrelin and adiponectin concentrations decrease (Cagnacci et al., 2002; Stachowiak et al., 2009).

The symptoms of menopausal syndrome are the most common reason for the application of menopausal hormonal therapy (MHT) in perimenopausal women. MHT aims to replace the natural ovarian hormonal activity through the administration of estrogen and/or progesterone in the minimum effective doses. The biological effects of hormone therapy depend on the types of estrogen and progesterone, and the doses of hormones. It also depends on the route of administration, with oral and transdermal MHT having significantly different effects (Pertyński and Stachowiak, 2006).

Transdermal MHT acts more favorably than oral MHT, because it affects the reduction of factor VII activity, reduces the levels of PAI-1 and E-selectin, and alters the lipid profile and carbohydrate balance. This results in a reduced risk of atherosclerosis and coronary heart disease (Menon and Vongpatanasin, 2006). Oral MHT significantly affects the digestive system in comparison to transdermal MHT, causing bloating, nausea, and vomiting. It also increases the cholesterol saturation of bile and contributes to the development of gallstones (Mueck and Seeger, 2005).

A review of the literature shows that there are few studies on the effects of MHT on parameters such as ghrelin or leptin levels in postmenopausal women. Results obtained by Kellokoski et al. (2005) and Lambrinoudaki et al. (2008) were divergent. The aim of this study was to evaluate the concentrations of the total and active form of ghrelin, and the leptin receptor, as well as lipoprotein(a) (Lp(a)) and PAI-1:Ag in postmenopausal women who received oral or transdermal MHT.

2. Materials and methods

2.1. Subjects

Women from all study groups were selected in the Outpatient Gynecology Centre of the University Hospital in Bydgoszcz, Poland (Ruszkowska et al., 2010a; 2010b; 2011). Written informed consent was obtained from each participant before entering the study. The study was permitted by the Bioethics Committee of the Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Poland (No. KB/305/2004).

The study was conducted on 76 healthy, non-smoking women, who were 1–2 years post-menopausal. Forty-six women aged from 44 to 58 years (mean age 52 years) used oral (26) or transdermal (20) MHT. MHT was taken daily during continuous treatment in the form of a composite preparation of estrogen and progesterone in different combinations. Twenty-six women from the study group used oral MHT [2 mg 17β-estradiol (E2) and 1 mg norethisterone acetate (NETA) (Kliogest, Novo Nordisk Pharma, Poland)] and 20 women used transdermal MHT [50 µg E2 and 170 µg NETA (SYSTEN® Conti, Janssen-Cilag, Warsaw, Poland)]. The subjects used MHT for 6–14 months. Reported climacteric symptoms, such as heavy and regular hot flushes with drenching sweat, were the main indicators for MHT (Ruszkowska et al., 2010a; 2010b; 2011).

The control group consisted of 30 healthy, nonsmoking, post-menopausal women, aged from 44 to 54 years (mean age 49 years), who did not use MHT.

Blood pressure (BP) and body mass index (BMI) were measured at the beginning of the study in all groups. The systolic blood pressure (SBP) was (120±19) mmHg, diastolic blood pressure (DBP) was (74±15) mmHg, and the BMI was (21.0±3.5) kg/m².

Women in all study groups had neither diabetes mellitus nor glucose intolerance. They had no prior thrombosis or systemic illnesses. None of them took any other medication that might have interfered with the coagulation system. All women included in the study had a complete gynecological examination, cytology smear, breast examination, and mammography (Ruszkowska et al., 2010a; 2010b; 2011).

Venous blood (4.5 ml) for tests of the leptin receptor, PAI-1:Ag, and Lp(a) was collected into cooled tubes (Becton Dickinson Vacutainer® System, Plymouth, UK) containing 0.13 mol/L trisodium citrate (the final blood-anticoagulant ratio was 9:1) after 30 min of rest between 7:30 and 9:30 am and after a 12-h overnight fast. The blood samples were immediately mixed and centrifuged at 3 000×g at 4 °C for 20 min. The obtained platelet-poor plasma was divided into 200 µl Eppendorf-type tubes and then samples were frozen at −20 °C (according to the manufacturer’s procedures) until assayed, but for no longer than three months. Some precautions were required in order to determine the concentration of the active form of ghrelin, because it was very unstable and labile in plasma. To prepare plasma samples, whole blood was drawn directly into a centrifuge tube that contained 500 U of aprotinin and 1.25 mg of ethylenediaminetetraacetic acid (EDTA)-2Na per 1 ml of whole blood. The tubes were mixed gently and then the blood samples were immediately centrifuged at 1 500×g for 15 min at 4 °C. Then, 100 µl of 1 mol/L HCl per 1 ml of collected plasma were added immediately. The obtained plasma was divided into 200 µl Eppendorf-type tubes and then the samples were frozen at ≤−40 °C until assayed. Blood for tests of total ghrelin was collected in a tube containing no anticoagulant (Becton Dickinson Vacutainer® 17490, Plymouth, UK). 4-(2-Aminoethyl)benzenesulfonyl fluoride (AEBSF) was then added to give a final concentration of 1 mg/ml, and the blood was allowed to clot at room temperature for 30 min. The serum was separated by centrifuging at 2 500×g for 15 min and kept at 4 °C. The serum was transferred to separate tubes and acidified with HCl to a final concentration of 0.05 mol/L and then the tubes were frozen at ≤−20 °C until assayed.

2.2. Metabolism and hemostasis assays

The concentrations of the active form of ghrelin and total ghrelin were measured by enzyme-linked immunosorbent assay (ELISA)—human ghrelin (active) ELISA and human ghrelin (total) ELISA (LINCO Research, St. Charles, Missouri, USA). The concentration of Lp(a) was measured by ELISA—lipoprotein(a) ELISA (ALPCO Diagnostics, Salem, USA); PAI-1:Ag was determined by ELISA—ASSERACHROM® PAI-1 (Diagnostica Stago, Asnieres, France). The concentration of the leptin receptor was measured by enzyme immunometric assay (EIA)—Leptin Receptor EIA (ALPCO Diagnostics, Salem, USA).

2.3. Statistical analysis

Statistical analysis was performed using the statistical program STATISTICA 9.1 StatSoft® software (StatSoft®>, Cracow, Poland). The Shapiro-Wilk test was used to assess the normality of distributions. For variables with normal distribution, arithmetic means (X) and standard deviations (SD) were determined. The median (Me), lower quartile (Q ₁), and upper quartile (Q ₃) were used for variables that were not normally distributed. The significance of differences between groups of normally distributed variables was analyzed using the Fisher-Snedecor analysis of variance (ANOVA), and for other variables the Kruskal-Wallis ANOVA test was used. P-values of <0.05 were considered statistically significant.

3. Results

Table 1 shows the concentrations of total ghrelin, the active form of ghrelin, leptin receptor, Lp(a), and PAI-1:Ag in women who received oral or transdermal MHT and in the control group. We observed a significantly higher concentration of total ghrelin and the active form of ghrelin in women who received transdermal MHT in comparison with the group of women who took oral MHT (P=0.0497; P=0.0220, respectively). We found also a significantly lower concentration of total ghrelin in women who took oral MHT compared with those in the control group (P=0.0286). Moreover, a higher concentration of PAI-1:Ag was found in the group of women who received transdermal MHT in comparison with those who used oral MHT and with the control group. The differences were statistically significant (P=0.0001 and P=0.0234, respectively).

Table 1.

Concentrations of assayed parameters in women who received oral or transdermal MHT and in the control group

Group	Total ghrelin (pg/ml)	Ghrelin active form (pg/ml)	Leptin receptor (ng/ml)	Lp(a) (mg/dl)	PAI-1:Ag (ng/ml)
Transdermal MHT (n=20)	134.06±35.90	82.25 (49.20/167.30)	27.63±8.93	6.68 (4.45/12.84)	58.25±20.37
Oral MHT (n=26)	102.80±48.33	48.20 (44.00/50.15)	32.90±9.61	4.40 (3.07/9.16)	31.96±10.85
Control (n=30)	139.64±56.78	50.55 (48.10/55.90)	26.78±10.63	4.48 (3.55/8.86)	41.82±26.10
P-value	0.0497*, 0.0286#	0.0220*	0.1012	0.4113	0.0234∆, 0.0001*

^* P<0.05, transdermal MHT vs. oral MHT

^# P<0.05, oral MHT vs. control group

^∆ P<0.05, transdermal MHT vs. control group

Values of studied parameters are shown as mean±SD or median (Q ₁/Q ₃)

Table 2 shows correlations between selected parameters. We found a significant negative correlation between the concentrations of total ghrelin and PAI-1:Ag (P=0.015) and a significant positive correlation between the concentrations of total ghrelin and leptin receptor (P=0.04), in women who received transdermal MHT.

Table 2.

Correlations between concentrations of ghrelin active form, total ghrelin and Lp(a), leptin receptor, PAI-1:Ag

Variable	Correlation coefficient
	Ghrelin active form		Total ghrelin
	Transdermal MHT	Oral MHT	Transdermal MHT	Oral MHT
Lp(a)	−0.2075	0.0975	−0.2564	−0.1505
	(P=0.477)	(P=0.658)	(P=0.376)	(P=0.493)
Leptin receptor	−0.1809	0.0860	0.5752	−0.2844
	(P=0.554)	(P=0.711)	(P=0.040*)	(P=0.212)
PAI-1:Ag	0.3516	−0.2356	−0.6344	−0.1393
	(P=0.218)	(P=0.268)	(P=0.015*)	(P=0.516)

^* P<0.05

4. Discussion

Menopause is an independent risk factor for polymetabolic syndrome. It is estimated that the risk of the polymetabolic syndrome increases for up to 14 years after menopause and then gradually decreases. Therapeutic procedure in perimenopausal women with metabolic syndrome involves the implementation of MHT. It has an undeniable impact on the balance of carbohydrate, and thus on insulin resistance. The use of estrogen during menopause leads to a reduction in the risk of visceral obesity, insulin resistance or type 2 diabetes. An appropriate choice of progestin is extremely important because improper use can lead to the abolition of the positive impact of estrogen on the balance of carbohydrate (Stachowiak et al., 2009).

In the present study, we obtained significantly higher levels of total ghrelin and the active form of ghrelin in women who received transdermal MHT compared with women who took oral MHT, and lower levels of total ghrelin in women who took oral MHT compared with the control group.

The main site of ghrelin synthesis is the gastric mucosa. Isomoto et al. (2005) observed that in the course of acute gastritis ghrelin levels increased, a response which is associated with an occurring inflammatory process. Because ghrelin has anti-inflammatory activity and it acts as a gastroprotective agent, it causes an increase in local blood flow and an increase in the production of prostaglandins (Konturek et al., 2004; Isomoto et al., 2005; Michalski et al., 2008). Ghrelin concentrations are significantly higher in lean individuals and in patients with chronic liver disease, celiac disease, bulimia or anorexia nervosa (Meier and Gressner, 2004; Michalski et al., 2008). The use of oral contraceptives containing estrogen and progesterone increases the concentration of ghrelin in women with severe malnutrition (Grinspoon et al., 2004). Conversely, lowering the concentration of ghrelin is associated with obesity and insulin resistance (Lambrinoudaki et al., 2008).

Results concerning a potential association between ghrelin levels and the route of administration of MHT in postmenopausal women have been conflicting (Lambrinoudaki et al., 2008). di Carlo et al. (2007) reported that women who received continuous transdermal hormone therapy (E2 at a dose of 50 mg/d and nomegestrol at 5 mg/d for 12 d/month), had higher levels of ghrelin compared with women who used oral estrogen-progesterone hormone therapy. Kellokoski et al. (2005) found a higher concentration of the active form of ghrelin in postmenopausal women who used oral estrogen therapy. Lambrinoudaki et al. (2008) did not observe statistically significant differences in circulating ghrelin concentrations between women who took oral estrogen therapy and those who had complex hormone therapy. However, Cagnacci et al. (2002) observed that postmenopausal women with a polimetabolic syndrome who used oral estrogen hormone therapy showed an increase in the level of leptin and a decrease in ghrelin concentration. This using transdermal estrogen hormone therapy showed a decrease in ghrelin levels but without any effect on leptin levels. This indicates a favorable effect of transdermal therapy on the polymetabolic syndrome factors in postmenopausal women.

The above results suggest that gastrointestinal bypass by using transdermal MHT has beneficial effects on the cardiovascular system in postmenopausal women. This in turn, may be linked to the anti-inflammatory and anti-atherosclerotic activities of ghrelin, which does not affect the adhesion or aggregation of platelets but is essential in reducing the formation of atherosclerotic plaque and thrombotic complications.

In the current study no statistically significant differences in leptin receptor concentrations were found, independently of the study group.

The soluble form of the leptin receptor affects leptin’s effect on food intake and body weight regulated ectodomain shedding of the membrane-spanning leptin receptor, which may constitute a new mechanism for regulating the biological function of leptin (Meier and Gressner, 2004). Patients with end-stage heart failure have elevated levels of leptin and its receptor. Leptin may participate in the process of catabolism and lead to cardiac cachexia in chronic heart failure (Christian Schulze et al., 2003).

Insulin resistance and visceral obesity are associated with low levels of soluble forms of the receptor and a low rate of binding of free leptin, independent of body fat (Meier and Gressner, 2004). Leptin reduces insulin secretion from pancreatic B-cells and participates in the inhibition of insulin-glucose synthesis in the liver. It is also a protein important in sexual maturation and maintenance of fertility, because its concentration in the blood affects the level of secreted hormones (estrogen) (Szumiło et al., 2007). Patients with lipodystrophy had significantly reduced levels of leptin, but hyperleptinemia is a basic feature of obesity. The BMI is the best predictor of leptin levels (Meier and Gressner, 2004). Leptin stimulates proliferation and inhibits apoptosis in normal and cancer cells (Nishi et al., 2005; Hoda et al., 2007).

The effect of MHT on leptin levels remains unclear. Several studies show no effect of MHT on leptin levels independent of the BMI (Lambrinoudaki et al., 2004; 2008). On the other hand, there are studies in which an increase in leptin was achieved after one month of MHT, after two months treatment with 2 mg of estradiol administered orally, and after six months of estrogen hormone therapy (Elbers et al., 1999; Konukoglu et al., 2000). These discrepancies may be explained by the indirect effect of MHT on lowering leptin levels (which reduces adipose tissue) in contrast to the direct effect of estrogen, which stimulates the secretion of leptin (Lambrinoudaki et al., 2008).

Analysis of the results obtained here allowed the establishment of a positive correlation between ghrelin and leptin receptor levels in women who used transdermal hormone therapy. Cummings and Foster (2003) suggested a correlation between ghrelin and leptin in the regulation of lipid changes.

A review of the literature shows that no similar correlation was found in postmenopausal women who took MHT. Bagnasco et al. (2002) obtained a correlation between changes in ghrelin levels and leptin levels. During fasting, plasma ghrelin levels increased whereas leptin concentrations decreased, but during food intake the situation was reversed. This observation can be explained by the fact that ghrelin, depending on the concentration, activates arcuate nucleus neurons and leptin is their inhibitor (Cummings and Foster, 2003). Meier and Gressner (2004) described a positive correlation between BMI and leptin concentrations and a negative correlation between BMI and ghrelin levels, depending on the population tested. Shiiya et al. (2002) found that the ghrelin concentration was negatively correlated with the BMI during fasting in groups of Japanese patients with and without diabetes. Tschöp et al. (2001) measured ghrelin levels in fasting adult Caucasians and Pima Indians, and obtained negative correlations between ghrelin and the amount of body fat and the concentrations of insulin and leptin.

Our results showed that women who used transdermal MHT had a higher concentration of PAI-1:Ag compared with the control group and the group of women who took oral MHT.

PAI-1 is the main inhibitor of the plasminogen conversion reaction to plasmin and is synthesized by vascular endothelial cells, smooth muscle cells of vessels, megakaryocytes, hepatocytes, and adipocytes. A significant amount of PAI-1 is stored in platelet granules, from which it is released under the influence of different activating agents. The concentration of PAI-1 is higher in men and it increases with age and with obesity. High concentrations of PAI-1 pose a risk of thrombotic cardiovascular disease, including coronary heart disease (Dobrovolsky and Titaeva, 2002).

A review of the available literature indicates that MHT stimulates the fibrinolytic system by increasing the concentrations of tissue plasminogen activator (t-PA) and lowering the levels of PAI-1. It was observed that estrogen administered in MHT directly affects the biosynthesis and secretion of PAI-1. However, the progestin component may reduce the beneficial effects of estrogen on the fibrinolytic system (Stachowiak et al., 2005). In the present study it was observed that the route of administration of MHT had a significant effect on the concentration of PAI-1:Ag in the blood. The higher levels of PAI-1:Ag found in women who used transdermal MHT compared with those who took oral MHT were consistent with the results of Post et al. (2003), Stachowiak et al. (2005), and Ruszkowska et al. (2010a). They demonstrated that oral MHT significantly reduces the levels of PAI-1 (up to 50%) but did not observe such a significant change associated with the use of transdermal MHT. Post et al. (2003) explained this observation as being due to the stimulation by t-PA of the release of PAI-1 in these women. Oral use of MHT results in stimulation of the synthesis of many proteins in the liver and in increased clearance in hepatocytes, not only t-PA but also PAI-1. Post et al. (2003) showed increased hepatic clearance in women who took oral MHT.

In the current study, a negative correlation between concentrations of PAI-1:Ag and ghrelin levels in women who used transdermal MHT was obtained. This indicates a favorable ratio between high concentrations of ghrelin and low levels of PAI-1:Ag. We can find no other study showing ghrelin’s effect on lowering the concentration of PAI-1:Ag. If it was true, it would indicate another beneficial action of ghrelin as increased fibrinolytic activity would reduce the risk of heart disease or thrombosis. It is known that ghrelin is an excellent cardiometabolic marker for predicting atherosclerosis in the elderly. It has also a beneficial effect on the cardiovascular system by reducing inflammation and lowering BP.

5. Conclusions

This study showed that women who used transdermal MHT had higher levels of total ghrelin than women who took oral MHT. This indicates a beneficial effect from using the transdermal route of administration of MHT. However, transdermal MHT is also associated with adverse effects with regard to the observed higher levels of PAI-1:Ag, which in turn, leads to a reduction in fibrinolytic activity.