J Endocrinol Invest (2014) 37:715–719 DOI 10.1007/s40618-014-0089-2
ORIGINAL ARTICLE
Plasma nesfatin-1 levels are increased in patients with polycystic ovary syndrome Esra Nur Ademoglu • Suheyla Gorar • Ayse Carlıoglu • Havva Yazıcı • Fatma Dilek Dellal • Zehra Berberoglu • Derya Akdeniz • Sema Uysal • Feridun Karakurt
Received: 10 December 2013 / Accepted: 30 April 2014 / Published online: 12 June 2014 Ó Italian Society of Endocrinology (SIE) 2014
Abstract Background Nesfatin-1 is a recently discovered neuropeptide derived from its precursor nucleobindin-2 (NUCB2) and has been implicated in the regulation of feeding and energy metabolism. It is located in the brain and also produced at the periphery and present in the plasma. However, its pathophysiological role in humans remains unknown. Polycystic ovary syndrome (PCOS) is commonly presented with obesity, insulin resistance, hyperandrogenemia and hirsutism. Aim To characterize serum nesfatin-1 levels in PCOS women and determine association of nesfatin-1 with metabolic parameters.
Materials and methods It is a cross-sectional study of 55 PCOS and 28 healthy women matched in age, in a university hospital setting. Anthropometric, hormonal, metabolic parameters and nesfatin-1 blood levels were determined. Results Nesfatin-1 levels were significantly higher in PCOS group compared with the controls 371.43 ± 2.50 versus 275.55 ± 1.74 pg/mL. Multivariate logistic regression analysis that contains: nesfatin-1, body mass index and homeostasis model assessment index revealed significant correlation of nesfatin-1 with the existence of PCOS (p \ 0.05). Conclusions Higher nesfatin-1 levels in PCOS women compared to control group may suggest a possibility that nesfatin-1 may play some role in the PCOS. Keywords syndrome
E. N. Ademoglu (&) S. Gorar F. D. Dellal Z. Berberoglu Department of Endocrinology and Metabolism, Ankara Education and Research Hospital, S¸ u¨kriye Mh, 06340 Sıhhıye, Ankara, Turkey e-mail:
[email protected] A. Carlıoglu F. Karakurt Department of Endocrinology and Metabolism, Faculty of Medicine, Turgut Ozal University, Ankara, Turkey H. Yazıcı Department of Internal Medicine, Erzurum Education and Research Hospital, Erzurum, Turkey D. Akdeniz Department of Internal Medicine, Faculty of Medicine, Turgut Ozal University, Ankara, Turkey S. Uysal Department of Biochemistry, Faculty of Medicine, Turgut Ozal University, Ankara, Turkey
Nesfatin-1 Obesity Polycystic ovary
Introduction Nucleobindin-2 (NUCB2)-derived nesfatin-1 has been recently identified in the satiety and control of energy homeostasis. It is a neuropeptide that has been implicated in the regulation of feeding and metabolism. Nesfatin-1/ NUCB2 is distributed in some brain regions, including the hypothalamic paraventricular nucleus (PVN), supraoptic nucleus, lateral hypothalamic area and the nucleus tractus solitarius [1, 2]. Nesfatin-1, an anorectic peptide first described in the central nervous system, is also expressed in the gastric endocrine cells, adipocytes and pancreatic beta cells. Intracerebroventricular administration of nesfatin-1 induces decreases in food intake and body weight and increases in sympathetic nerve activity and mean arterial
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pressure [3]. Pancreatic beta cells producing insulin colocalize with nesfatin/NUCB2 in the islets of both mice and rats, suggesting the possible role of nesfatin-1 in the regulation of insulin secretion from pancreatic beta cells [4]. In the light of the fact that some of the brain-gut peptides, including glucagon-like peptide-1(GLP-1), pituitary adenylate cyclase-activating polypeptide (PCAP) and ghrelin, regulate both the brain and pancreatic islet functions, it seems that nesfatin-1 may have a role in the regulation of insulin release [5, 6]. Recently, it was shown that nesfatin-1 promotes Ca2? influx through L-type channels and enhances glucose-induced insulin secretion in mouse islet b-cells [7, 8]. Polycystic ovary syndrome, characterized by chronic anovulation and hyperandrogenism, is the most common endocrine disorder of women of reproductive age while the etiology of the syndrome remains enigmatic. PCOS is a multi-factorial disease resulting from the dysfunction of different systems because of the fact that patients suffering from the disease have different hormonal and metabolic patterns [9]. Insulin resistance (IR) is a common feature of polycystic ovary syndrome, which is found in approximately 30 % of lean women and in majority of obese women and contributes to development of hyperinsulinemia, which drives hyperandrogenemia in these patients [10, 11]. Obesity, present in approximately one-half of patients with PCOS, often is the initial complaint of PCOS. The close relationship of obesity and IR with PCOS, furthermore the role of nesfatin-1 in food intake, insulin secretion and energy metabolism made us to investigate levels of nesfatin-1 in PCOS patients. We compared nesfatin-1 levels in a PCOS group with those in healthy individuals. We also assessed the association between nesfatin-1 and several metabolic parameters in these subjects (Fig. 1).
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Materials and methods Subjects The prospective study group was composed of 55 pregnant women who had recently been diagnosed with PCOS (mean age 25.1 years) at the Outpatient Clinic of the Turgut Ozal University Faculty of Medicine, Endocrinology Department, and 28 healthy women matched in terms of age have formed the control group (mean age 26.2 years). Plasma nesfatin-1 levels were measured in both groups. The diagnosis of PCOS was based on the 2003 ESHRE/ ASRM diagnostic criteria. Patients who had at least two of the following conditions were accepted as having PCOS: clinical and/or biochemical hyperandrogenemia, oligo or anovulation, PCOS morphology in transabdominal ultrasonography. Chronic anovulation was identified by requiring fewer than eight cycles per year and serum progesterone levels (PRG) of less than 9.54 nmol/L. Hyperandrogenemia was identified as total testosterone levels being above the 95th percentile of the levels detected in the group of normal menstruating. PCOS morphology was defined as the presence of 12 or more follicles of size 2–9 mm on the ovary or having unilateral ovary volume of at least 10 cm3 by ultrasonography [12]. Ovarian volume was calculated by the formula (0.5 9 ovarian length 9 width 9 thickness). A synacthen test was conducted in each women with a basal 17-hydroxyprogesterone (17OHP) plasma level [3 nmol/L. Control group was composed of healthy women with regular menstrual periods who had no hyperandrogenemia, hirsutism or acne. All control subjects were euthyroid, normoprolactinemic and normoandrogenemic. Exclusion criteria for the study were known cardiovascular disease, renal or liver impairment, diabetes mellitus, hypertension, neoplasms, preeclampsia, hyperprolactinemia, Cushing’s syndrome, nonclassical congenital adrenal hyperplasia, acute or chronic inflammatory disease known. None of the subjects were taking any drugs or smoking. The body mass index (BMI) was calculated for all participants [BMI: body weight (kg)/ square height (m2)]. The ethical committee of Turgut Ozal University Hospital approved the study. A written informed consent was obtained from all participants. Assays
Fig. 1 Nesfatin-1 concentrations in the PCOS groups
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Blood sampling was conducted after one night of fasting on the third to fifth day of the follicular phase or at any time in anovulatory women with PRG levels \9.54 nmol/L for fasting serum insulin (FSI), fasting blood glucose (FBG), hemoglobin A1c (HbA1c), total testosterone (TT), DHEAS,
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follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), 17-OHP, cortisol and ACTH. The samples were centrifuged immediately, and the serums were stored at -80 °C until the time of analysis for nesfatin-1. Serum nesfatin-1 concentrations were analyzed with the ELISA kits from USCN Life Science Instruments (Wuhan, China). In this assay system, the intra-assay and inter-assay coefficient of variation were always below 10 %. The assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific for human NES1 has been precoated onto a microplate. A competitive inhibition reaction was launched between the biotinlabeled human NES1 and unlabeled human NES1 (standards or samples) with the precoated antibody specific for human NES1. After incubation, the unbound conjugate was washed off. Next, avidin conjugated to horseradish peroxidase (HRP) was added to each microplate well and incubated. The amount of bound HRP conjugate was inversely proportional to the concentration of NES1 in the sample. After addition of the substrate solution, the intensity of color developed was inversely proportional to the concentration of NES1 in the sample. Serum concentrations of 17-OHP were measured with a commercially available kit (Adaltis S.r.l, Milano, Italy), which is a microplate chemiluminescence assay for quantitative determination. Homeostasis model assessment index (HOMA-IR) for IR was calculated for each patient using the formula: fasting plasma glucose (mmol/L) 9 fasting insulin (mIU/mL)/22.5.
717 Table 1 Demographical characteristics and biochemical values of controls and PCOS
All statistical analyses were carried out using the statistical package SPSS version 15.0 (SPSS, Inc., Chiago, IL). Shapiro–Wilk test was used to check the normality of the distributions of all the continuous variables. The normally distributed variables were analyzed with the Student’s t test, and the variables that did not show a normal distribution were compared with Mann–Whitney U test. Bivariate correlations between nesfatin-1 and insulin, HOMA-IR, BMI, glucose in the PCOS group were calculated and analyzed by the Spearman correlation test. Multivariate logistic regression analysis that contains: nesfatin-1, BMI and HOMA-IR, was performed to examine association of nesfatin-1 with the existence of PCOS. p values less than 0.05 were considered statistically significant for all statistical analyses. The data for continuous variables were presented as mean ± SD.
Results Demographical characteristics and biochemical values of PCOS patients and healthy controls are shown in Table 1.
PCOS (n = 55) 25.1 ± 5.6
Age (years)
26.2 ± 4.9
BMI (kg/m2)
21.0 ± 2.8
27.4 ± 6.8**
FBG (mmol/L) HOMA-IR
4.78 ± 0.39 1.72 ± 1.14
4.86 ± 0.49* 2.80 ± 2.00*
Total cholesterol (mmol/L)
0.04 ± 0.78
0.02 ± 0.90
TG (mmol/L)
0.81 ± 0.37
0.99 ± 0.40
HDL (mmol/L)
1.47 ± 0.42
1.34 ± 0.46
LDL (mmol/L)
2.30 ± 0.62
2.45 ± 0.76
* p \ 0.05; ** p \ 0.001, between PCOS and controls
Table 2 Hormonal values of controls and patients with PCOS
Nesfatin-1 (pg/mL) FSI (pmol/L)
Controls (n = 28)
PCOS (n = 55)
275.55 ± 1.74
371.43 ± 2.50*
55.56 ± 37.22
81.53 ± 50.0*
Testosterone (ng/dL)
0.61 ± 0.06
1.31 ± 0.09**
DHEAS (ng/mL)
6.28 ± 3.47
8.22 ± 4.02
FSH (IU/L)
6.67 ± 1.62
5.75 ± 2.28
LH (IU/L)
6.44 ± 4.39
6.44 ± 3.46
271.06 ± 30.14 2.57 ± 0.85
169.05 ± 14.05 2.73 ± 114.79
3.48 ± 0.60
3.12 ± 2.42
140.87 ± 88.70
233.91 ± 166.52*
E2 (pmol/L) PRG (nmol/L) 17-OHP (nmol/L) PRL (nmol/L)
Statistical analysis
Controls (n = 28)
* p \ 0.05; ** p \ 0.001, between PCOS and controls
The patients with PCOS and the control group were similar in terms of mean age. Serum levels of testosterone, PRL, FSI and HOMA-IR in patients with PCOS were significantly elevated compared with the controls (p \ 0.05), whereas FSH, LH, progesterone, cortisol and DHEAS were not significantly different (Tables 1, 2). Additionally, BMI of the PCOS group was higher than the controls (p \ 0.05). Serum nesfatin-1 concentrations were significantly higher in the PCOS group compared with the control group (371.43 ± 2.50 vs. 275.55 ± 1.74 pg/mL) (p \ 0.05). Multivariate logistic regression analysis that contains: nesfatin-1, BMI and HOMA-IR revealed significant correlation of nesfatin-1 with the existence of PCOS (p \ 0.05) (Table 3). When the Spearman correlation was computed using the nesfatin-1 as the dependent variable, a significant negative correlation of nesfatin-1 with BMI (r = -0.326, p \ 0.05) was found. Nesfatin-1 did not correlate with fasting glucose, nor with HOMA-IR (Table 4).
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Table 3 Multivariate logistic regression analysis that contains HOMA-IR, BMI, nesfatin-1 OR
95 % CI
p value
HOMA-IR
1,685
0,833–3,406
0.146
BMI
1,516
1,134–2,026
0.005
Nesfatin-1
1,006
1,001–1,011
0.020
Dependent variable: PCOS; independent variable: nesfatin-1, HOMAIR, BMI
Table 4 The correlation analyses of nesfatin-1 with some parameters in PCOS subjects Parameters
r value
p value
BMI
-0.326
0.021*
HOMA-IR FSI FBG
0.031
0.841
-0.069
0.653
0.120
0.418
* p \ 0.05 is statistically significant
Discussion In this study, we found that serum nesfatin-1 levels are significantly higher in women with PCOS compared to healthy individuals, and there is an association of nesfatin1 with the existence of PCOS independently of IR and high BMI. Polycystic ovary syndrome has lifelong implications with increased risk for infertility, metabolic syndrome, type 2 diabetes mellitus and possibly cardiovascular disease. Nesfatin-1 is a newly discovered hormone that is involved in the regulation of nutritional status and food intake. There are limited data about the clinical importance of this protein. Recent studies demonstrated that nesfatin-1 reduces food intake in rodents when administered peripherally. The expression of nesfatin-1 both in central nervous system and in peripheral tissues including pancreatic beta cells suggests the possible involvement of nesfatin-1 in the regulation of insulin secretion from pancreatic beta cells [13]. Based on the data that either PCOS or nesfatin-1 has a close relationship with BMI, obesity and IR, one would expect that nesfatin-1 and PCOS might have a relationship as well. In the literature, there is only one study about the nesfatin-1 levels in PCOS patients, where the circulating nesfatin-1 levels were found to be lower in patients than in controls. In that study, Deniz et al. pointed out that they did not know whether the decrease in nesfatin-1 levels is mediated through IR or is the result of other metabolic factors. They hypothesized that lower nesfatin-1 levels may play a role in the development of PCOS [14]. However, in contrast to that study, we have demonstrated that serum
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nesfatin-1 is elevated in patients with PCOS. The clinical importance of nesfatin-1 in PCOS has not been established in previous studies, and little data are currently present in the literature; hence, the discrepancy between the two studies might require further investigation. The difference may be attributed to the differences in the study design, including patient selection and experimental conditions. Tsuchiya et al. established a highly sensitive and specific ELISA for investigating circulating nesfatin-1 levels, and they reported that fasting nesfatin-1 concentrations were significantly lower in subjects with high BMI compared to nonobese subjects. Li et al. investigated fasting plasma levels of nesfatin-1 in patients with type 1 and type 2 diabetes mellitus. They showed that fasting nesfatin-1 was significantly lower in T2 DM patients compared to healthy subjects and T1 DM patients. They pointed that while the significance of this result is unclear, the reduction in fasting nesfatin-1 may be due to one of the appetiterelated hormones involved in diabetic hyperphagia [16] and that nesfatin-1/NUCB2 release from islets is impaired in Goto-Kakizaki rats, a type 2 model diabetic model characterized with impaired insulin secretion and visceral fat accumulation [17]. Similarly, in another study, maternal serum nesfatin concentrations were measured in patients with gestational diabetes mellitus, and it has been reported that maternal plasma nesfatin-1 levels are significantly lower in gestational diabetes mellitus compared to healthy pregnant individuals [18]. In contrast to these studies, Zhang et al. [13] demonstrated that serum nesfatin-1 was found to be increased in patients with newly diagnosed type 2 diabetes mellitus. It was shown that plasma nesfatin1 correlated positively with BMI, HbA1c, fasting blood glucose, fasting plasma insulin and HOMA-IR. Furthermore, in another study, it was demonstrated that NUCB2 mRNA expression in the pancreatic islets is markedly increased in diet-induced obese mice associated with hyperinsulinemia and IR [19]. Similarly, Ramanjaneya et al. [20] found increased nesfatin-1 levels in obese states in both rodents and humans in their study. In the present study, we found that nesfatin-1 concentrations had a negative association with obesity and BMI while there was not any correlation with HOMA-IR and fasting glucose. Deniz et al. revealed a negative correlation between nesfatin-1 and BMI which was in agreement with our study. Furthermore, they showed a negative correlation between nesfatin-1 and fasting blood glucose and HOMAIR in disagreement with our study. Similarly, Tsuchiya et al. showed a significant negative correlation between nesfatin-1 concentrations and BMI and blood fasting glucose. In this study, nesfatin-1 concentrations were lower in obese patients when compared with nonobese patients. However, some studies revealed that nesfatin-1 levels measured with standard commercial kits showed positive
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correlation with BMI. For example, Saldanha et al. showed such a positive correlation in patients with diabetes mellitus. They hypothesized that nesfatin-1 may be important for homeostasis of energy metabolism and decreased levels may lead to decreased energy expenditure in vivo [13–21]. Previous data about association of nesfatin-1 and BMI were rather controversial. There is a close relationship between obesity and PCOS. Many studies have shown that most obese people demonstrated marked leptin resistance. Leptin is one of the most important anorexigenic proteins, which is positively correlated with BMI. Increased circulating leptin fails to suppress appetite in obese patients, indicating the existence of leptin resistance [22]. Although it has been shown that nesfatin-1 is able to cross the blood–brain barrier to induce satiety, postreceptor signaling pathways for nesfatin-1 in central nervous system and beta cells are unknown and it remains to be further elucidated. Taking into account the fact that nesfatin-1 is an anorexigenic protein, it may be possible to speculate that nesfatin-1 resistance may exist in PCOS like leptin resistance in obesity because of impaired receptor and postreceptor signaling in target tissues. Alternatively, another reason for higher nesfatin-1 levels in PCOS group may be a compensatory upregulation of the adipokine to counteract obesity. The novelty of this study is that we demonstrate increased nesfatin-1 levels in patients with PCOS for the first time. Our finding raises a possibility that nesfatin-1 may play a significant role in the PCOS. However, larger scale and molecular studies are needed to further investigate this topic. Conflict of interest
All authors have no conflict of interest.
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