Archives of Gynaecology and Women Health

Abstract

Introduction: Menopause is a stage in which women present physiological and metabolic changes, including visceral fat accumulation or lipid profile alterations, responsible for an increased risk of developing cardiovascular diseases, metabolic syndrome, or osteoporosis. Because adipokines, through their corresponding receptors, participate in different metabolic functions, a change in the serum concentration of these proteins is often associated with cardiometabolic impairment. Lipocalin-2 (LCN2) is an adipokine synthesized in several organs whose secretion increases when an inflammatory or infectious process occurs. Estrogen down-regulates lipocalin-2 expression within gonadal adipose tissue, while in bone tissue, this adipokine regulates energy homeostasis in osteoblasts. Considering the latter, as well as the scarce information regarding the role lipocalin-2 plays during menopause, we considered it important to measure the serum levels of lipocalin-2 in females in the pre and postmenopausal stages to assess if there is a change in lipocalin-2 serum levels between these two reproductive stages and also if there is a correlation between this change and those corresponding to other metabolic parameters.

Methods: We conducted a prospective cross-sectional study of 79 healthy women, comprising 33 in the premenopausal stage and 46 in the postmenopausal stage, who were not receiving hormonal replacement therapy. To analyze all the biochemical parameters, sera from all subjects were obtained after centrifugation of 5 ml of venous blood samples drawn after an 8-12-hour fasting period. We quantified all biochemical and anthropological parameters according to previously reported methodology. We assessed the circulating levels of adipokines using competitive electro-immunoassay kits.

Results: The average age of participants was 52 ± 0.7 years. The mean ± SEM for the Body Mass Index (BMI) within the two groups was 29.5 ± 0.90 kg/m2. Fasting glucose levels were within the normal range in both groups. The rest of the biochemical parameters were higher than the normal range. Osteocalcin levels were higher in postmenopausal women (6.1 ± 0.46 ng/mL) than those reported in women in the premenopausal stage (5.2 ± 0.88 ng/mL) (P ≤ 0.03). In the premenopausal group, the lipocalin-2 circulating concentration was 28 ± 3.4 ng/mL, whereas postmenopausal women presented a reduction in lipocalin-2 circulating levels (18 ± 2.7 ng/mL; P≤ 0.003). Regarding estradiol, the average value in the premenopausal group was 99 ± 18 pg/mL and 19 ± 0.23 pg/mL in the postmenopausal group (P≤ 0.0001). In addition, during post menopause, lipocalin-2 correlated positively with estrogen (r=0.35, P≤ 0.04).

Discussion: We found that osteocalcin serum levels increased significantly in postmenopausal women. Moreover, in this group, data analysis showed a statistically significant positive correlation between lipocalin-2 serum concentration and estrogen levels. The latter was unexpected because we initially hypothesized an increment of lipocalin-2 levels due to a decline in estrogen. These results may not be due to a direct molecular interaction among the variables studied but rather a consequence of the metabolic state within the postmenopausal group.

Abbreviations

ADIPO = Adiponectin

BMI Bone Mass Index

ER = Estrogen receptor

ESR1 (Estrogen receptor Alpha gene 1)

ELISA = Electroimmunoassay

FSH= Follicle Stimulating Hormone

HDL= High density lipoprotein

HOMA-IR= Homeostasis model assessment index-insulin resistance

IR= Insulin resistance

KDa= Kilodaltons

LCN2= Lipocalin-2

LDL= Low density lipoprotein

LXRβ= Liver X receptor beta

MMP-9= Matrix metalloprotease-9

OCN= Osteocalcin

PPARɤ= Peroxisome proliferator-activated receptor gamma

TC= Total cholesterol

TG= Triglycerides

VFA= Visceral fat

Introduction

Menopause is a reproductive transition that spans from the late reproductive years through perimenopause to the early and late menopausal stages. It is characterized by irregular menstrual cycles, vasomotor symptoms, changes in body fat composition, and metabolic profile, all of which are caused by a decline in estrogen synthesis [1-2]. Among these symptoms, the increase in abdominal obesity and visceral adiposity is directly associated with an increased risk of developing cardiometabolic diseases, osteoporosis, and certain malignancies, partly due to dysregulation of adipokines within different organs [3]. Lipocalin-2 (LCN2) was initially identified in human neutrophils by its covalent binding to the matrix metalloprotease 9 (MMP-9) [4-5]. This 25 kDa glycoprotein is present in several organs, including those involved in the cardiometabolic system [6-7]. LCN2 is an adipokine whose secretion increases when an inflammatory or infectious process occurs [8-9]. This glycoprotein is involved in glucose and lipid metabolism, which is dysregulated in conditions such as type 2 diabetes mellitus, fatty liver disease, or obesity [10]. It is worth mentioning that lipocalin-2 presents a sex-specific expression within adipose tissue. Female mice present a higher expression of the corresponding protein in inguinal white adipose tissue than male mice; on the contrary, male mice exhibit higher levels of lipocalin-2 in perigonadal white adipose tissue [10-11]. In addition, the authors of this study demonstrated that in female mice deficient for lipocalin-2, the serum estradiol levels and estrogen alpha receptor expression and activity were drastically reduced in various metabolic tissues compared to the levels exhibited by control females. Also, mice Lcn2 (-/-) had a decrease in the expression of the aromatase enzyme in the adipose tissues analyzed. Moreover, the reduction of the three parameters studied in mice null for lipocalin-2 was more significant when the authors provided a high-fat diet and with age. Also, the authors reported that Lcn2 (-/-) mice showed decreased expression of various transcription factors involved in lipid metabolism (PPARγ, LXRβ, and LDL-R). Another study reported that the human and murine genes for lipocalin-2 (LCN2 and Lcn2, respectively) contain an estrogen response site in their promoter regions, suggesting that estrogen regulates the transcription of both genes [12]. Likewise, Drew and colleagues (2015) demonstrated that the estrogen-estrogen receptor signaling pathway is involved in the downregulation of lipocalin-2 expression in gonadal adipose tissue [13]. A few years later, Krishnan et al. (2019) analyzed the expression and regulation profile of Lcn2 in the adipose tissue and liver from obese animal models, finding that only in female adipose tissue, estrogen regulates the expression of Lcn2, and its upregulation leads to metabolic complications, such as mitochondrial dysfunction and inflammation [14]. Recently, lipocalin 2 has emerged as a bone-derived protein. Two studies have demonstrated that LCN2 is expressed at levels 10 times higher in osteoblasts than in other organs, acting as a negative modulator of bone formation. Moreover, these authors proposed that its release into the blood is sensitive to nutritional signals through the melanocortin-4 receptor, establishing its role in appetite regulation and glucose homeostasis [14-15]. Considering all the above and taking into account that lipocalin 2 is a protein involved in energy homeostasis whose expression in adipose tissue is regulated by estrogen in a sex-specific manner, we wanted to analyze if the lack of estrogen in postmenopausal women leads to an increment in the serum levels of lipocalin-2 and also assess if there is a correlation between this change and those corresponding to other metabolic parameters.

Methods

We conducted a prospective cross-sectional study in a cohort of 79 healthy women attending the Endocrine Service of the Siglo XXI National Medical Center, who accepted to enroll in this study and signed the informed consent after the protocol's authorization, following the guidelines established by the Official Mexican Standard for employing human samples in research protocols (3601-2018) and the Declaration of Helsinki [16]. Thirty-three women in the premenopausal stage and 46 in the postmenopausal stage were included in the study, aged 45–56 years (Mean ± SEM= 52 ± 0.7) (Table 1). The menopausal diagnosis was based on the development of amenorrhea >1 year, FSH levels >30 mU/mL, and estradiol <30 pg/mL. Exclusion criteria were as follows: women receiving hormonal replacement therapy, presence of diabetes, cardiovascular disease, hepatic or renal dysfunction, any infection, or malignancy that could lead to an inflammatory process. We measured anthropometric and biochemical parameters according to previously reported methodology [17]. Increments of measurements for weight and height were 0.1 kg and 0.01 m, respectively. Body Mass Index (BMI) was calculated as weight (kg) divided by height (m) squared. Obesity was defined according to a BMI of 30 kg/ m2 or more, and overweight according to a BMI of 25 kg/m2 or more, but less than 30 kg/m2. Waist circumference was measured in centimeters using a tape measure placed at mid-waist. In order to quantify the concentration of all the biochemical parameters, we obtained 5 ml of venous blood from each subject after an 8-12 hour fasting period. Each blood sample was centrifuged at 857 x g for 10 minutes at 10 °C (Beckman Gs-15R, GMI, Inc., Ramsey, Min., USA) and immediately stored at –70 °C for subsequent assays. Glucose and lipid profiles were assessed as previously described [17]. Intra- and inter-assay coefficients of variation for all measurements were < 7%. Insulin resistance (IR) was estimated using the homeostasis model assessment index-insulin resistance (HOMA-IR). HOMA-IR is fasting glucose (mg/dL) multiplied by fasting insulin (µU/ml), divided by 405. Dyslipidemia was defined as any of the following: total cholesterol > 200 mg/dL, HDL < 40 mg/dL, LDL > 130 mg/dL, or triglycerides > 150 mg/dL.

Assessment of Adipokines' Concentrations

All adipokine circulating levels were assessed using competitive immunoassay ELISA kits: Lipocalin-2 (R&D Systems, Minneapolis, MN, USA), osteocalcin (AVIVA Systems Biology, San Diego, CA, USA), and adiponectin (Human Adiponectin/Acrp30 Catalog Numbers: DY1065-05). The assays and the standard curves were performed in duplicate, following the instructions of the corresponding protocols. The absorbance of each sample was measured using a multidetector spectrophotometer (Victor 3 1420, PerkinElmer, Turku, Finland). The intra-assay variation coefficient was <10%. Values were expressed in the case of lipocalin-2 in ng/mL for adiponectin (ADIPO) in µg/mL.

Statistical Analysis

Data is presented as mean ± standard error (SEM). We performed nonparametric comparisons for TG, HDL, total cholesterol (TC), estradiol, osteocalcin, adiponectin, and lipocalin-2 levels. We also performed an average distribution analysis. Categorical variables were analyzed using the chi-square test and continuous variables, such as the student's t-test (for normally distributed variables) and the Mann–Whitney U test (for non-normally distributed variables). Additionally, we performed a multivariate regression model and a Spearman correlation analysis to assess which variables were most effective in predicting outcomes. A p-value ≤ 0.05 was considered statistically significant. We used GraphPad Prism version 10 for all statistical analyses.

Results

Table 1 summarizes the results for all anthropometrical and biochemical parameters. Regarding metabolic parameters, fasting glucose levels were within the normal range in both groups (85.5 ± 2.7 mg/dL). Triglyceride concentrations were 147 ± 11 mg/dL in premenopausal women and 154 ± 11 mg/dL in postmenopausal women. Serum total cholesterol concentration was 214 ± 6.4 mg/dl and 230 ± 8 mg/dL, respectively. HDL levels were 53 ± 1.8 mg/dL. LDL levels were 134 ± 5.9 mg/dL in the premenopausal group and 146 ± 7.2 mg/dL in the postmenopausal group. HOMA-IR index was 3.5 ± 0.36 and 4 ± 0.32, respectively. Additionally, most women presented high levels of visceral fat. The premenopausal group had a value of 134 ± 11 cm², and the postmenopausal group had a value of 140 ± 11 cm².

Data on adiponectin levels were as follows: 11 ± 1.2 µg/mL in the first group and 13 ± 1.1 µg/mL in the second group. Average estradiol levels were 99 ± 18 pg/mL and 19 ± 0.23 pg/mL, respectively (P= 0.0001). Mineral bone density values were 4.5 ± 0.33 kg in premenopausal women and 3.6 ± 0.07 kg (P= 0.0009) in the postmenopausal group (Table 1).

Data are expressed as mean ± standard error (SEM). Values were compared between groups employing a Mann-Whitney test for independent samples. Significance *P<0.05; **P<0.01; ***P<0.0001. n= number of samples, BMI= body mass index, HDL= high density lipoprotein, LDL=low density lipoprotein, HOMA-IR= homeostasis model assessment-insulin resistance, VFA= visceral fat, OCN = osteocalcin, LCN2= lipocalin-2, ADIPO= adiponectin, E2= estradiol, TC=Total cholesterol.

Table1: Anthropometrical and biochemical data of premenopausal and postmenopausal women. Regarding lipocalin-2 serum concentration, in premenopausal women was 28 ± 3.4 ng/mL, whereas in postmenopausal women presented a statistically significant reduction of this adipokine circulating levels (18 ± 2.7 ng/mL; P= 0.003). Os-teocalcin levels were higher in postmenopausal women than those reported in women in the premenopausal stage (6.1 ± 0.46 ng/mL vs 5.2 ± 0.88 ng/mL, p= 0.03) (Figure 1).

A serum concentration of lipocalin-2 (LCN2) and B Serum concentration of osteocalcin in pre and postmenopausal women. Data are expressed as mean ± standard error (SEM) in ng/ml. Values were compared between groups employing a Mann-Whitney test for independent samples. Statistical significance **P<0.003 and *P<0.03, respectively.

Figure 1: Serum concentration of Lipocalin 2 (LCN2) and Osteocalcin (OCN) in pre- and postmenopausal women.

The Spearman rank correlation test indicated that during pre- menopause, there is a strong positive correlation between ADIPO and HDL (r= 0.41, P ≤ 0.03), between BMI and HO- MA-IR (r= 0.52 p ≤ 0.002), between BMI and bone mineral density (r= 0.91 P ≤ 0.0005), between bone mineral density and VFA (r= 0.77 P ≤ 0.01), bone mineral density with LDL (r= 0.68 P≤ 0.03), and between VFA with HOMA-IR (r= 0.56 P ≤ 0.0007). On the contrary, we obtained a negative correlation between ADIPO and HOMA-IR (r=-0.40, P≤ 0.04), between BMI and HDL (r=-0.45, P≤ 0.01), and between HDL and VFA (r=-0.41, P ≤ 0.02) (Figure 2).

Spearman's rank correlation test among lipocalin-2 (LCN2) and all the variables studied in premenopausal women. The bar on the left has 2 colors, blue, which indicates a positive correlation between the variables analyzed, and red, which indicates a negative correlation. The greater the intensity of the color, the greater the degree of correlation between the variables.

Figure 2: Spearman rank correlation test among all the variables studied in premenopausal women

During postmenopause, lipocalin-2 correlates positively with estrogen (r=0.35, P≤ 0.04); we also found a positive correlation between BMI and HOMA-IR (r=0.38, P ≤ 0.01) and between HOMA-IR and VFA (r=0.45, P≤ 0.02). Adiponec-tin levels correlated negatively with HOMA-IR (r=-0.48, P≤ 0.005) (Figure 3).

Spearman rank correlation test among lipocalin-2 (LCN2) and all the variables studied in postmenopausal women. The bar on the left has 2 colors, blue, which indicates a positive correlation between the variables analyzed, and red, which indicates a negative correlation. The greater the intensity of the color, the greater the degree of correlation between the variables.

Figure 3: Spearman rank correlation test among all the variables studied in postmenopausal women

Discussion

During menopause, most women present an alteration in their body fat composition and dysregulation of several metabolic traits that increase the risk for the development of cardiometabolic diseases. Lipocalin-2 is a protein involved in numerous physiological processes, regulated by estrogen in a sex and depot-specific manner, whose circulating concentrations increase in the presence of acute pathological events. In addition, studies have demonstrated that bone releases lipocalin-2 ten times more than adipose tissue to participate in energy homeostasis and appetite regulation [18]. Considering all the above, we hypothesized that due to estrogen decline, the circulating concentration of lipocalin-2 would increase in postmenopausal women, and this increment would also be associated with corresponding levels of osteocalcin. Our study showed a statistically significant difference between pre- and postmenopausal osteocalcin and lipocalin-2 levels. We did not observe any correlation between lipocalin-2 circulating levels and those of osteocalcin. However, we found a positive correlation between lipocalin-2 and estrogen in postmenopausal women. In 2021, Moon and colleagues conducted a cross-sectional study on 5,896 menopausal women, divided into two groups based on their metabolic status [19]. The study found that OCN levels were lower in menopausal women with metabolic syndrome compared to those without metabolic syndrome. Nevertheless, and in agreement with our results, the postmenopausal group (with and without metabolic syndrome), analyzed by Moon and co-authors, presented higher levels of osteocalcin (20.21 ± 7.79) than those observed in the premenopausal group (14.08 ± 5.13). The latter could be explained as a defense mechanism to increase osteoblast activity and counteract osteoclast-mediated bone turnover during post menopause when the risk of osteoporosis increases [20-21]. On the contrary, other studies have reported that OCN concentration was higher in individuals with metabolic syndrome [22-23]. In this regard, it has been proposed that the elevation of OCN may be a compensatory mechanism secondary to the elevation of insulin to improve glucose metabolism [24]. Even though osteocalcin and lipocalin-2 are synthesized in osteoblasts, and both proteins are involved in metabolism and energy expenditure, we found no correlation between serum lipocalin-2 and osteocalcin in premenopausal or postmenopausal women. In agreement with our results, Mauritzi and colleagues (2021) reported that lipocalin-2 correlated with age and the Dickkopf WNT Signaling Pathway Inhibitor 1 (DKK1) serum concentrations, but they found no correlation between this adipokine and any bone turnover biomarker analyzed in postmenopausal women, suggesting that lipocalin-2 mediates indirectly the relationship between age and DKK1 [25]. Regarding lipocalin-2, in addition to visceral fat accumulation, estrogen decline increases the synthesis and secretion of the follicle-stimulating hormone, which can elevate the circulating levels of different adipokines [26]. Considering the latter and the fact that metabolic dysregulation is more significant in postmenopausal women, we expected a higher lipocalin-2 serum concentration in this group. Nevertheless, lipocalin-2 levels were lower in the postmenopausal group than in premenopausal women (18 ± 2.7 ng/ml vs 28 ± 3.4 ng/ml) (Figure 1). Our results contradict several studies that indicate lipocalin-2 as a pro-inflammatory protein whose levels increase to counteract metabolic dysregulation [7-9-27]. However, we and others observed that subjects with several years having type 2 diabetes mellitus presented statistically significantly lower levels of LCN2 than the ones observed in the healthy population, which led us to conclude that this adipokine does not act as an anti-inflammatory protein once metabolic dysfunction exceeds a specific point [28-29]. Therefore, it makes sense that in our study, the serum concentration of lipocalin-2 decreased in women who had passed several years since menopause. Because lipocalin-2, estrogen, and estrogen receptor alpha regulation are intertwined, the primary purpose of this study was to analyze if such a correlation occurred between the serum levels of LCN2 and those corresponding to estrogen in premenopausal and postmenopausal women. Within the postmenopausal group, we found a strong positive correlation between lipocalin-2 and estrogen levels (r=0.35, P≤ 0.04) (Figure 4). The latter coincides with the results presented by Guo and colleagues (2012), which described that Lcn2 deficient mice presented lower levels of serum estradiol and lower expression of proteins involved in lipid metabolism within the gonadal adipose tissue, leading to a lipid metabolism dysregulation and the generation of an inflammatory state [11]. The relevance of this study resides in its description of a link between the null concentration of this adipokine and a lipid metabolism disruption within a specific adipose depot, which is associated with an increase in fat mass during menopause. However, as with our study, these authors were unable to identify the physiological relationship between lipocalin-2 and the associated traits. To determine the role of Lcn2 in cardiometabolic complications, Krishnan and colleagues (2019) employed an adenoviral vector to specifically achieve lipocalin-2 expression in white adipose tissue and liver of mice with an Lcn2-null background and under a high-fat diet [14]. They found that lipocalin-2 expression led to metabolic complications, mitochondrial dysfunction, and inflammation only within the white adipose tissue of female mice. Also, the increment of lipocalin-2 expression caused the downregulation of the lipocalin-2-specific receptor, megalin, and the reduction of the expression of the estrogen receptor alpha gene (Esr1). Because estrogen receptor alpha binds directly to the Lcn2 promoter to inhibit its genetic expression within the adipose tissue, to these authors, it was unexpected to find an ER alpha downregulation by lipocalin-2 within the female adipose tissue. Therefore, they also concluded that lipocalin-2 regulates obesity in a tissue and sex-specific manner, which remains to be determined. Unfortunately, one of our limitations is that we do not analyze the expression profile of lipocalin-2, the estrogen-alpha receptor, or megalin in the visceral adipose tissue of both groups of women; therefore, we cannot infer that there is a molecular process within adipose tissue that could be interfering with the decrease in the concentration of lipocalin-2 in the serum of postmenopausal women.

Conclusions

This study found that lipocalin-2 serum levels decreased significantly in postmenopausal women. Moreover, data analysis revealed a statistically significant positive correlation between the lipocalin-2 serum concentration in this group and estrogen levels. The latter may not be due to a direct molecular interaction but rather a consequence of the metabolic state within the postmenopausal group.

Declarations

Funding Sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data Availability:

Research data is available upon request. To request the data, please contact the corresponding author.

Acknowledgements

We thank M. Sc Leticia Damasio Santana from the Endocrine Research Unit, Siglo XXI National Medical Center, Mexican Social Security Institute, for her kind assistance with the immunoassay.

Acknowledgements

We thank M. Sc. Leticia Damasio Santana from the Endocrine Research Unit, Siglo XXI National Medical Center, Mexican Social Security Institute, for her kind assistance with the immunoassay.

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