Abstract
Context
Insulin resistance has been detected in a majority of patients with polycystic ovary syndrome (PCOS). Elevated neprilysin levels are associated with insulin resistance.
Objective
The present study aims to investigate plasma neprilysin and its relationship with endocrine and metabolic characteristics in patients with PCOS.
Subjects and Methods
Thirty-five premenopausal PCOS patients and 35 healthy volunteers of similar age were included in the study. Demographic characteristics, biochemical and hormonal findings and also plasma neprilysin levels were determined in these patients and healthy controls.
Results
In our study, HOMA-IR values were significantly higher in PCOS patients (3.3 ± 1.8) compared with the controls [(1.6 ± 1), p<0.01]. Plasma neprilysin levels were significantly higher in the PCOS group compared to the control group (1502.1 ± 1641.2 vs. 764.6 ± 562.6 pg/mL). There was no difference in plasma neprilysin levels when PCOS patients were classified as overweight-obesity (BMI≥25kg/m2) or non-obesity (BMI<25kg/m2).
Conclusion
Our findings revealed significantly higher levels for plasma neprilysin and HOMA-IR values in PCOS patients when compared to controls. No significant differences were noted between obese PCOS patients and non-obese PCOS patients in terms of plasma neprilysin levels.
Keywords: polycystic ovary syndrome, neprilysin, obesity, insulin resistance
Introduction
Neprilysin (NEP, neutral endopeptidase) is a plasma membrane protein expressed from pancreatic islet cells and nonislet tissues such as mesenteric adipose tissue, endothelial, epithelial, smooth muscle cells, and fibroblasts (1). Neprilysin leads to degradation and inhibition of many small vasoactive peptides such as endothelin, bradykinin, atrial (ANP) and, brain (BNP) natriuretic peptide, angiotensin I and II (2). Neprilysin also may play a role in regulating insulin receptor internalization in pancreatic β cells (3). Metabolically altered states including obesity and type 2 diabetes upregulate neprilysin (4).
Neprilysin cleaves active GLP-1 at six sides. This leads to the loss of GLP-1’s ability to bind its receptor (5, 6). Inhibition of neprilysin may increase active GLP-1 levels via preventing proteolysis of GLP-1 and/or reducing dipeptidyl peptidase-4 (DPP-4) activity. Moreover, except for the GLP-1 dependent mechanisms, neprilysin inhibition improved glycemia via blocking bradykinin degradation (7).
Polycystic ovary syndrome (PCOS) is the most prevalent endocrinopathy affecting women at reproductive age and associated with reproductive, metabolic, and psychological dysfunction. In addition to hyperandrogenaemia, hirsutism, oligo or amenorrhea, and anovulation, PCOS women exhibit reduced insulin sensitivity and β-cell dysfunction. Furthermore, abnormal adipokines are secreted from the adipose tissue (8-9).
Despite a long history of studies, the etiology and pathophysiology of PCOS remain to be ascertained (10). However, it is known that dysregulation of insulin action is closely related to the pathogenesis of PCOS (11). The present study aims to investigate insulin resistance, plasma neprilysin, and its relationship with endocrine and metabolic characteristics of PCOS patients.
Materials and Methods
Study population
Thirty-five PCOS patients and 35 healthy volunteers of similar age who had followed-up at our center were included in the study. The criteria for inclusion in the PCOS group were: to be diagnosed as PCOS based on 2003 Rotterdam revised criteria (12): (i) anovulation or irregular ovulation, (ii) clinical and/or biochemical hyperandrogenism, (iii) polycystic ovaries. We confirmed two of the three diagnostic criteria provided that other causes that may lead to a similar finding are excluded. The control group consisted of 35 healthy volunteers who were not diagnosed with PCOS and did not report menstrual irregularities and did not have clinical and laboratory hyperandrogenism. Written consent of the study subjects and ethics committee approval from our center were obtained.
Study parameters
Body weight, height, waist circumference, hip circumference, systolic blood pressure (SBP) (mmHg), diastolic blood pressure (DBP) (mmHg), pulse (bpm) measurements and body fat ratios of all patients and healthy participants included in our study were analyzed. Body mass index (BMI) and fat percentage was measured on a daily calibrated scale and recorded (Tanita, Body Fat Monitor, Tanita Corporation,Tokyo, Japan). Waist circumference was measured at a midpoint between the bottom of the rib cage and the top of the iliac crest at the end of exhalation. Hip circumference was measured at the level of maximal gluteal protrusion. Waist to hip ratio was calculated by dividing the waist circumference to hip circumference measurement. Polycystic ovarian morphology was confirmed in patients by ultrasound. Hirsutism was evaluated using the Ferriman–Gallwey score, and menstrual disturbances (none, oligomenorrhea, amenorrhea) were evaluated based on medical history in all subjects.
Biochemical analysis
Blood glucose, insulin, homeostasis model assessment of insulin resistance (HOMA-IR) formula: fasting plasma glucose (mmol/L) x fasting serum insulin (mU/L)/22.5, liver function tests, lipid profile (total cholesterol, HDL-cholesterol, LDL-cholesterol and triglyceride) and plasma neprilysin levels were performed after fasting for 8-10 hours in all patients and healthy volunteers. Follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin, total testosterone, 17 hydroxyprogesterone (17OHP), dihydroepiandosterone-sulfate (DHEA-S) and androstenedione levels were evaluated. Concentrations of neprilysin were measured in plasma using ELISA (Adipo Bioscience, Santa Clara, USA).
Statistical Analysis
Statistical analysis was made using computer software SPSS version 21.0 (SPSS Inc.). Demographic, basal characteristics and neprilysin levels are summarized with mean and standard deviation for continuous variables, frequency and percentages for categorical variables. Statistical analysis was performed using Chi-square test for comparison of the categorical data, for comparison of the continuous data between the study and control groups Student’s t-test was used for normally distributed variables and Mann–Whitney U variance analysis tests was used for not normally distributed variables. Spearman’s correlation coefficients were calculated to assess the associations between variables. The significance level of p-value less than 0.05 was considered as statistically significant.
Results
A total of 70 patients (35 patients with PCOS [mean age: 26.8 ± 4.3 years] and 35 control patients [mean age: 27.6±3.2 years], p=0.39) who were followed up in the endocrinology and metabolic diseases outpatient clinic of our center were included in the study. Data on patient anthropometric measurements and vital signs are summarized in table 1. Bodyweight was 74.8 ± 18.1 kg in the PCOS group and 61.4 ± 13.3 kg in the control group. Bodyweight was significantly higher in PCOS group (p=0.001). Waist-to-hip ratio and body fat ratio were 0.84 ± 0.10 and 31.7 ± 9.3% in the PCOS group, 0.74 ± 0.05 and 25.2 ± 7.8% in the control group, respectively. The difference between waist-hip ratio and fat ratio was statistically significant between the two groups (p<0.001 and p=0.004, respectively). While SBP was significantly higher in the PCOS group (109.1 ± 7.4 vs. 112.8 ± 6.6 mmHg, p=0.04), diastolic blood pressure and heart rate were similar in both groups (Table 1).
Table 1.
Comparison of demographic data of the patients according to the groups
| PCOS group (n=35) | Control group (n=35) | p value | |
|---|---|---|---|
| Height (cm) | 162.0±6.1 | 164.4±6.4 | 0.13 |
| Weight (kg) | 74.8±18.1 | 61.4±13.3 | 0.001 |
| BMI (kg/m2) | 28.5±6.9 | 22.8±5.3 | <0.001 |
| Waist-hip ratio | 0.84±0.10 | 0.74±0.05 | <0.001 |
| BFR (%) | 31.7±9.3 | 25.2±7.8 | 0.004 |
| SBP (mmHg) | 109.1±7.4 | 112.8±6.6 | 0.04 |
| DBP (mmHg) | 68.0±7.1 | 67.3±12.2 | 0.63 |
| Heart rate (pulse/min) | 79.7±5.4 | 78.9±5.4 | 0.56 |
PCOS: polycystic ovary syndrome, BMI: Body mass index, BFR: Body fat ratio, SBP: Systolic blood pressure, DBP: Diastolic blood pressure, min: minute.
The patients included in our study were compared in terms of hormonal parameters. There was no statistically significant difference between total testosterone, DHEA-S and 17OHP levels between PCOS and control group. Androstenedione levels were significantly higher in PCOS group compared to the control group (4.5 ± 1.6 vs. 3.2 ± 1.3 ng/mL, p=0.001) (Table 2).
Table 2.
Comparison of hormone data of the patients according to the groups
| PCOS group (n=35) | Control group (n=35) | p value | |
|---|---|---|---|
| T-testosterone (ng/mL) | 1.1±0.4 | 1.1±0.2 | 0.77 |
| DHEA-S (µg/dL) | 326.2±134.5 | 288.6±93.0 | 0.31 |
| Androstenedione (ng/mL) | 4.5±1.6 | 3.2±1.3 | 0.001 |
| Prolactin (ng/mL) | 18.8±13.6 | 15.1±7.1 | 0.28 |
| 17OHP (ng/mL) | 1.3±0.7 | 1.0±0.5 | 0.27 |
| FSH (mIU/mL) | 4.7±1.1 | 5.3±2.4 | 0.97 |
| LH (mIU/mL) | 8.4±5.7 | 7.6±7.4 | 0.16 |
PCOS: polycystic ovary syndrome, T-Testosterone: Total testosterone, DHEA-S: Dihydroepiandosterone-sulfate, 17OHP: 17 hydroxyprogesterone, FSH: Follicular stimulating hormone, LH: luteinizing hormone.
When the groups were evaluated in terms of laboratory data, fasting blood glucose (FBG), total cholesterol, LDL-cholesterol and triglyceride levels were significantly higher and HDL-cholesterol levels were lower in the PCOS group (p=0.002, p=0.03, p=0.008, p=0.03 and p=0.005, respectively) (Table 3). While there were no difference in aspartate transaminase (AST) levels from liver enzymes, alanine transaminase (ALT) levels were higher in PCOS group (19.7 ± 14.1 vs. 12.8 ± 6.3 U/L). Serum fasting insulin and HOMA-IR were significantly higher in PCOS group (14.4 ± 7.3 mIU/L and 3.3 ± 1.8 in the PCOS group, 8.2 ± 4.8 mIU/L and 1.6 ± 1.0 in the control group, p<0.01, respectively). Plasma neprilysin levels were significantly higher in PCOS group compared to control group (1502.1 ± 1641.2 pg/mL vs. 764.6 ± 562.6 pg/mL, p=0.027) (Table 3). There was no difference in plasma neprilysin levels when PCOS patients were classified as overweight-obesity (BMI≥25kg/m2) or normal weight (BMI<25kg/m2). No correlation of plasma neprilysin levels with BMI, waist circumference, waist-hip ratio, plasma glucose and HOMA-IR was noted (Table 4).
Table 3.
Comparison of laboratory data of the patients according to the groups
| PCOS group (n=35) | Control group (n=35) | p value | |
|---|---|---|---|
| FBG (mg/dL) | 88.7±9.0 | 81.9±8.0 | 0.002 |
| TC (mg/dL) | 189.2±36.2 | 165.7±38.7 | 0.03 |
| HDL-C (mg/dL) | 46.6±6.9 | 54.1±11.1 | 0.005 |
| LDL-C (mg/dL) | 119.8±32.9 | 97.2±25.8 | 0.008 |
| Triglyceride (mg/dL) | 113.5±57.3 | 85.7±39.5 | 0.03 |
| AST (U/L) | 18.2±6.6 | 15.9±3.5 | 0.09 |
| ALT (U/L) | 19.7±14.1 | 12.8±6.3 | 0.02 |
| Insulin (mIU/L) | 14.4±7.3 | 8.2±4.8 | <0.001 |
| HOMA-IR | 3.3±1.8 | 1.6±1.0 | <0.001 |
| Neprilysin (pg/mL) | 1502.1±1641.2 | 764.6±562.6 | 0.027 |
PCOS: polycystic ovary syndrome, FBG: Fasting blood glucose, TC: Total cholesterol, HDL-C: High density lipoprotein-cholesterol, LDL-C: Low density lipoprotein-cholesterol, AST: aspartate transaminase, ALT: alanine transaminase, HOMA-IR: Homeostasis model assessment.
Table 4.
Correlation between metabolic parameters and neprilysin levels in the patients
| Spearman’s rho | BMI | WC | WHR | FPG | HOMA-IR | |
|---|---|---|---|---|---|---|
| Neprilysin (PCOS group) | Correlation Coeffficient | -0.44 | -0.11 | 0.06 | -0.22 | 0.03 |
| Sig. (2-tailed) | 0.80 | 0.52 | 0.72 | 0.21 | 0.86 | |
| Neprilysin (Control group) | Correlation Coeffficient | 0.03 | 0.005 | 0.17 | 0.26 | -0.08 |
| Sig. (2-tailed) | 0.86 | 0.97 | 0.31 | 0.13 | 0.65 |
PCOS: polycystic ovary syndrome, BMI: Body mass index, WC: Waist circumference, WHR: Weist-hip ratio, FPG: Fasting plasma glucose, HOMA-IR: Homeostasis model assessment.
Discussion
Polycystic ovary syndrome is one of the most frequent reproductive endocrine disease of women with a prevalence estimated to be 5-10% in reproductive age (13). Approximately 50-70% of PCOS patients have insulin resistance and 30% of them are obese (14). Extensive data support that PCOS patients suffer from metabolic disorders including obesity, insulin resistance and type 2 diabetes that consist of common features of PCOS but not diagnostic criteria of PCOS (13, 15). Many studies have also shown that PCOS patients are at an increased risk of hypertension (16). In our study, the mean body weight of the PCOS patients included in the study was 74.8 ± 18.1 kg and it was found to be statistically higher when PCOS patients were compared with healthy controls of similar age group. Similarly, serum insulin and HOMA-IR levels were significantly higher in PCOS patients compared to the control group. Higher serum levels of total cholesterol, LDL-cholesterol and triglycerides and SBP, and lower levels of HDL-cholesterol in our PCOS patients seem in line with the indication of higher rates for metabolic syndrome in PCOS patients than in controls (13, 14). These findings showed us, metabolic syndrome criteria were higher in patients with PCOS in this study.
It has been found that reducing neprilysin activity improves insulin secretion and sensitivity (4). It is thought that neprilysin is expressed from adipocytes and plays a role in blood pressure regulation and cardiometabolic risk (2). In a study evaluating 318 male patients, neprilysin activity was found to be correlated with insulin resistance and metabolic syndrome features (2). Plasma neprilysin level was also correlated with body mass index. As the metabolic syndrome criteria increased, plasma neprilysin levels also increased progressively (2). Treatment with neprilysin inhibitors resulted with increased insulin sensitivity in rats (17). Neprilysin degrades and inactivates GLP-1 and pharmacological inhibition of neprilysin provides higher circulating levels of GLP-1 (18). This finding suggests that neprilysin could affect insulin secretion and insulin sensitivity. Combination of neprilysin and DPP-4 inhibition has also been found more effective at maintaining active GLP-1 levels than inhibition of DPP-4 alone (19). Studies also showed that neprilysin inhibition is associated with protection against impaired β-cell function (18). Neprilysin is expressed in non-islet tissues such as mesenteric fat and endothelium, and upregulated in the increase of free fatty acids. Elevated levels of chronic free fatty acids cause defects in many pathways during insulin production and secretion. Neprilysin is also expressed in pancreatic islet cells and is suggested to play a role in islet cell dysfunction associated with free fatty acids (20, 21). In our study, plasma neprilysin levels were significantly higher in the PCOS group compared to the control group (1502.1 ± 1641.2 vs. 764.6 ± 562.6 mmol/L). High levels of neprilysin in patients with PCOS may use to be an indicator of impaired insulin metabolism and increased cardiometabolic risk.
Since insulin resistance plays a major role in the pathogenesis of PCOS, insulin sensitizing agents especially metformin has been used in treating PCOS. Our findings revealed significantly higher levels for plasma insulin and HOMA-IR values in PCOS patients when compared to controls, which seem in agreement with the statement that excess body weight in women with PCOS may accelerate progression toward diabetes by exacerbating both insulin resistance and compensatory enhanced insulin response (22, 23). Because it has been found that reducing neprilysin activity improves insulin secretion, and sensitivity and treatment with neprilysin inhibitors resulted with increased insulin sensitivity in rats, the results of our study may be a guide for future studies evaluating neprilysin inhibitors for treating PCOS.
It has been demonstrated that elevated neprilysin levels are associated with increased BMI and HOMA-IR (2). In a study conducted by Ghanim et al., it was also found that after weight loss following Roux-N-Y gastric bypass, plasma neprilysin concentration was significantly decreased (24). Although plasma neprilysin levels were higher in patients with PCOS patients, similar to HOMA-IR, there was no correlation between HOMA-IR and plasma neprilysin levels in our study. In this study, metabolic syndrome parameters were found to be high in patients with PCOS. High plasma neprilysin levels in patients with PCOS may be associated with metabolic syndrome parameters and insulin resistance in these patients. The high plasma neprilysin level in patients with PCOS was thought to be related to the presence of metabolic syndrome and insulin resistance in these patients. When the patients with PCOS were evaluated as overweight-obese and normal weight according to BMI, plasma neprilysin levels were similar in both groups. This finding suggests that plasma neprilysin levels are associated with metabolic syndrome parameters independently of obesity.
The most important limitation of our study is that it is a cross-sectional study. The other important limitation is the small number of patients included to the study. On the other hand, to the best of our knowledge, there is no study evaluating neprilysin levels in PCOS patients. We think that the result of the present study may provide evidence to perform new studies in the future investigating the significance neprilysin in diagnosis of PCOS, as well as treatment of neprilysin inhibitors in treating PCOS. We can not say that plasma neprilysin levels can be used as a diagnostic parameter in the clinical approach for PCOS with the available data. However, with the results we obtained, we can say that neprilysin may have an important role in PCOS and may be a candidate to be used as a criterion in the diagnosis of PCOS.
In conclusion, the present study has shown that plasma neprilysin levels, metabolic syndrome parameters, and insulin resistance were high in patients with PCOS. Therefore, levels of plasma neprilysin may be useful in determining insulin resistance and detecting insulin resistance-related metabolic problems regardless of obesity in patients with PCOS.
Conflict of interest
The authors declare that they have no conflict of interest.
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