The Incretin Effect and Secretion in Obese and Lean Women with Polycystic Ovary Syndrome: A Pilot Study

Abstract

Background:

Insulin resistance is considered to play an important role in the pathogenesis of polycystic ovary syndrome (PCOS) and in the progression to type 2 diabetes. Recent reports concentrate on a possible relationship between incretin secretion and beta-cell function in PCOS. The aim of the present study is to investigate the incretin effect in obese and lean women with PCOS.

Methods:

Twenty women with PCOS and ten age-matched healthy women were recruited in the study. The oral glucose tolerance test (OGTT) and isoglycemic test were carried out on each participant after an overnight fast at 2-weeks interval. Plasma levels of insulin, glucose, C-peptide, glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) were assayed.

Results:

Obese women with PCOS demonstrated lower GIP concentrations (area under the curve [AUC]) in response to OGTT compared to the control group. The incretin effect was found significantly augmented in the obese women with PCOS compared to controls. This finding remained robust in the subgroup analysis including only body mass index (BMI)-matched healthy women.

Conclusions:

Increased insulinotropic effect could counteract the blunted GIP response to OGTT in obese women with PCOS. It is suggested that the pathology of PCOS may also include impaired activity of the enteroinsular axis.

Introduction

Polycystic ovary syndrome (PCOS) is a common endocrine disorder, affecting nearly 10% of reproductive-aged women.^1,2 It is characterized by a clustering of oligo/anovulation, clinical or biochemical hyperandrogenism, and polycystic ovaries. The pathophysiology of PCOS is complex, involving the hypothalamus-pituitary-ovarian axis, ovarian theca cell hyperplasia, insulin resistance (IR), and a multitude of other cytokine-driven and adipocyte-driven factors.³ Approximately 50% of women with PCOS are overweight or obese, and many metabolic abnormalities, such as dyslipidemia and hyperglycemia, are inconsistently present in the majority of these patients.^4,5 Women with PCOS also demonstrate increased risk of type 2 diabetes (T2D),^6,7 increased risk of gestational diabetes mellitus (GDM),⁸ and accumulation of cardiovascular disease (CVD) risk factors.^9,10 IR is considered to play an important role in both the pathogenesis of PCOS and the progression to T2D. Notwithstanding the known effects of excess adiposity on insulin sensitivity, evidence suggests that women with PCOS demonstrate an intrinsic form of IR that is independent and additive to obesity.¹¹ Therefore, current data address insulin-sensitizing drugs as a promising therapeutic option for chronic treatment of PCOS.¹² However, whether PCOS-related hyperinsulinemia is a direct consequence of IR solely or the net effect of an interaction between IR and altered incretin effect is not yet fully elucidated.

In normal subjects, 65%−70% of the glucose-dependent insulin secretion is attributed to the incretin effect (IE) of the gastrointestinal hormones, glucose-dependent insulinotropic peptide (GIP) and glucagon like peptide-1 (GLP-1).^13,14 Impaired secretion and insulinotropic activity of incretins have been described in T2D and obesity. In T2D, GLP-1 secretion is blunted, whereas GIP is normal yet lacking insulinotropic activity.¹⁵ On the other hand, obese patients demonstrate increased GIP and insulin levels¹⁶ but attenuated GLP-1 response to an oral glucose tolerance test, (OGTT),¹⁷ which returns to normal after successful gastric bypass.¹⁸

Several studies have focused on investigation of the relationship between incretin secretion and beta-cell function in PCOS. An older study by Gama et al.¹⁹ failed to detect a significant difference in GIP and GLP-1 levels between people with PCOS and healthy individuals. However, more recent data provide conflicting results on the incretin secretion pattern in PCOS patients in relation to body mass index (BMI).^20,21 Despite this inconsistency in the results regarding incretin response, a possible contribution to hyperinsulinemia observed in PCOS remains intriguing.

Therefore, the aim of the present study is to evaluate the insulinotropic action of GIP and GLP-1 in PCOS patients after controlling for the confounding effect of IR and obesity.

Participants and Methods

Twenty women with PCOS (10 obese and 10 lean) were recruited from the University outpatient clinic. Women with a normal BMI (<25) were classified as lean (group 1), and overweight and obese women with a BMI of at least 25 formed study group 2. Ten healthy, nonhirsute premenopausal, eumenorrheic women, matched for BMI with the women with PCOS, were recruited as controls. Controls had no personal or family history of hirsutism or endocrine disorders, and they were currently taking no medication. Diagnosis of the PCOS was based on the presence of chronic anovulation (fewer than six cycles in 12 months) and clinical or laboratory hyperandrogenemia. Other common causes of hyperandrogenemia and anovulation (prolactinoma, congenital adrenal hyperplasia, Cushing syndrome, and virilizing ovarian or adrenal tumors) were excluded in accordance with the revised criteria proposed in 2003 by the Rotterdam European Society for Human Reproduction/American Society for Reproductive Medicine (ESHRE/ASRM) sponsored PCOS Consensus Workshop Group.²² Hirsutism was evaluated by the Ferriman-Gallway score, and women with a score of at least 8 were classified as having hirsutism. All women reported no use of oral contraception (OC) or other medication that could alter glucose and insulin metabolism within the last 3 months. Women with T2D, family history of T2D, impaired glucose tolerance, or other chronic diseases known to impair glucose metabolism were excluded from the study. Informed consent was obtained from all participants, and the study was approved by the Ethics Committee of the Institution.

Study protocol

In the screening visit, basal fasting blood samples were drawn between the third and the fifth day of a regular menstrual cycle or after administration of progestagen for 10 days for amenorrhea. Serum and plasma concentrations of glucose, insulin, total testosterone, and dehydroepiandrosterone sulfate (DHEA-S) were measured. Insulin sensitivity was assessed in fasting conditions with the quantitative insulin sensitivity index (QUICKI), using an area under the curve (AUC)-derived threshold of 0.31 standardized for the prediction of abnormal glucose tolerance in Mediterranean women with PCOS, as has been described.²³ Women with impaired sensitivity were excluded from the study.

Two tests, the OGTT and isoglycemic test, were carried out on each participant after an overnight fast at 2-week interval.

Oral glucose tolerance test

All participants reported to our laboratory between 8.00 am and 8.30 am and underwent a 3-hour OGTT (75 grams). Blood samples were drawn at −15, 0, 5, 10, 15, 20, 30, 60, 90, 120, 150, and 180 minutes. Participants were advised to avoid eating, drinking other than water, smoking, and walking in the course of the test.

Isoglycemic test

At the second visit, a 3-hour isoglycemic test was performed. Catheters were inserted in the right and left antecubital veins for blood sampling and infusion. The plasma glucose profile that had been derived from the OGTT was reproduced by a variable intravenous glucose (20% dextrose) infusion using an ad hoc-developed algorithm. Venous blood was drawn again at −15, 0, 5, 10, 15, 20, 30, 60, 90, 120, 150, and 180 minutes. In both tests, blood was collected into chilled tubes with ethylenediaminetetraacetic acid (EDTA) (6.75 mM, final concentration) and 0.6 trypsin-inhibiting units (TIU) of aprotinin (600∼750 ballebrein-inhibiting units [KIU] of aprotinin) per 1 mL of blood sample. Plasma and serum were separated by ultracentrifugation and stored in −70°C until analysis.

Incretin effect calculation

The IE was estimated by the ratio of the difference between oral and intravenous AUC to oral AUC through the formula: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*}{\rm IE}=(\hbox{oral AUC}-\hbox{IV AUC})*100/ \hbox{oral AUC} \end{align*} \end{document}

Assay measurements

Serum testosterone levels (ng/dL) were measured by an electrochemiluminescence immunoassay (ECLIA) (Cobas, Roche) on a Modular Analytics E170 analyzer (Roche) according to the manufacturer's instructions. The minimum detectable concentration is 2 ng/dL, and the within-run and total assay coefficients of variation (CVs) are 1.1%−4.6% and 1.7%−7.4%, respectively. Limited cross-reactions are detected with androstendione (0.91%) and DHEA-S (0.01%).

Serum DHEA-S levels (μg/dL) were measured using an ECLIA on a Modular Analytics E170 analyzer according to the manufacturer's instructions. The minimum detectable concentration is 0.10 μg/dL, and the within-run and total assay CVs are 1.7%−2.8% and 2.4%−4.7%, respectively. Limited cross-reactions are detected with androstendione (0.399%), DHEA (0.178%), androsterone (0.033%), testosterone (0.033%) and aldosterone (0.008%).

Serum sex hormone-bending globulin (SHBG) levels (nmol/L) were measured by an enzyme-linked immunosorbent assay (ELISA) that used as enzyme conjugate mouse monoclonal SHBG antibody conjugated with horseradish peroxidase (HRP) (Biosource Europe). The minimum detectable concentration is 0.2 nmol/L, and the intraassay and interassay CVs are 3.0%−8.6% and 7.2%−11.6%, respectively.

Plasma glucose levels (mg/dL) were measured by a glucose oxidase colorimetric technique on an automated analyzer (Targa-Menarini).

Serum insulin levels (μIU/mL) were measured in duplicate by an enzyme-amplified sensitivity immunoassay (INS-EASIA) (Biosource) according to the manufacturer's instructions. The sensitivity of the assay is 0.17 μIU/mL (95% confidence level [CI]). The interassay and intraassay CVs are between 8.1%−9.0% and 4.8%−6.0%, respectively.

The serum levels of C-peptide (ng/mL) were measured in duplicate by ELISA (IBL) according to the manufacturer's instructions. The sensitivity of the assay is 0.064 ng/mL (95% CI). The intraassay and interassay CVs are between 5.13% and 6.70% and 8.38% and 9.33%, respectively.

Total GIP (ng/mL) and GLP-1 (ng/mL) levels were measured by nonradioactive ELISA kits (Phoenix Pharmaceuticals). The sensitivity of the assays is 0.11 ng/mL (95% CI). The intraassay and interassay CVs are <5% and <14%, respectively. The assays have 100% cross-reactivity to the intact forms of human GIP (1-42) and GLP-1 (7-36) and the inactive forms GIP (3-42) and GLP-1 (9-36) after N-terminal cleavage by the enzyme dipeptidyl peptidase-4 (DPP-4). Therefore, DPP-4 inhibitor was not used during preparation of the blood samples.

Statistical analyses

Data were described as mean±standard deviation (SD). AUCs were calculated by the trapezoidal rule. To detect a deviation from normality, the Shapiro-Wilk test was used for all variables. Analysis of variance (ANOVA) or Kruskas-Wallis test were used for comparisons between groups at baseline. Subgroup analyses (group 1, group 2, and BMI-matched control subgroup) included comparisons with the BMI-corresponding PCOS groups and were held with the use of Student's t test or Mann-Whitney test, where appropriate. A mixed model ANOVA for repeated measures analyzed differences in time course between groups during oral glucose tolerance test (OGTT). The Greenhouse-Geisser correction was used when the Mauchly test provided evidence that the sphericity assumption had been violated. Post hoc analysis Bonferroni adjustment was performed for statistically significant differences between groups. Two-tailed statistical significance was set at 0.05. Statistical analysis was performed using SPSS for Windows, version 17.0 and State/MP 10.0 (Statutory LP 4905).

Results

The clinical, biochemical, and hormonal parameters of the participants are summarized in Table 1. Age, insulin sensitivity (QUICKI), SHBG, and baseline GIP values did not differ between the women with PCOS and the control group. However, the basal GLP-1 concentrations were significantly lower in the obese PCOS women compared to both the control group (p=0.023) and the lean PCOS group (p=0.02) (Table 1). Total testosterone and DHEAS were significantly higher in women with PCOS (both group 1 and group 2) compared to controls.

Table 1.

Baseline Characteristics of Control, Lean Polycystic Ovary Syndrome, and Obese Polycystic Ovary Syndrome Groups

	Controls	Group 1 (Lean)	Group 2 (Obese)
Number	10	10	10
Age	25.3 (4.99)	22.2 (3.12)	23.5 (3.37)
BMI	25.03 (5.94)^a	21.1 (2.46)^b	31.23 (5.1)^a,b
QUICKI^d	0.43 (0.06)	0.51 (0.24)	0.39 (0.07)
GIP (ng/mL)^d,e	0.41 (0.23)	1.55 (1.08)	1.59 (0.43)
GLP-1 (ng/mL)^d,e	0.38 (0.08)^a	0.37 (0.28)	0.18 (0.1)^a
Testosterone (ng/dL)	46.62 (4.62)^a	62.6 (6.31)	72.1(9.09)^b
SHBG (nmol/L)^d	39.15 (15.78)	28.0 (32.25)	32.0 (29.0)
DHEA-S (μg/dL)	226.4 (3.89)^c	399.4 (31.83)^c	393.64 (40.86)^c

Data are presented as mean values (standard deviations) unless otherwise stated.

Statisticallly significant difference at the level of 0.025 (Bonferroni correction) ^abetween obese PCOS and control group, ^bbetween obese and lean PCOS groups, and ^cbetween control group and both obese and lean PCOS groups.

Median (interquartile range).

Morning fasting.

BMI, body mass index; DHEA-S, dehydroepiandrosterone sulfate; GIP, glucose-dependent insulinotropic peptide; GLP-1, glucagon-like peptide-1; QUICKI, quantitative index; SHBG, sex hormone-binding globulin.

No significant difference between groups was detected in fasting glucose, insulin, and C-peptide levels. Similarly, there was no significant difference between the control group and those with PCOS in the AUC for insulin (Mann-Whitney U-test, p=1.0), C-peptide (Mann-Whitney U-test, p=0.8), and GLP-1 (Mann-Whitney U-test, p=0.271) in response to OGTT. AUC values for insulin, C-peptide, GIP, and GLP-1 are presented in Table 2. However, GIP response differed between the PCOS and control groups (Mann-Whitney U-test, p=0.018). Group 2 demonstrated lower GIP concentrations (AUC) in response to OGTT compared to the control group (Mann-Whitney U-test, p=0.029) (Fig. 1). This finding remained robust in the subgroup analysis (including only obese controls, p=0.01). When exploring responses to the isoglycemic test (sparing IE on insulin secretion), no difference was detected between the PCOS and control groups in the AUCs for insulin (p=0.312), C-peptide (p=0.187), GIP (p=0.312), and GLP-1 (p=0.135).

FIG. 1.

Glucose-dependent insulinotropic peptide (GIP) response (area under the curve [AUC], ng/mL/min) to the oral glucose tolerance test (OGTT) in control women and lean and overweight/obese women with polycystic ovary syndrome (PCOS).

Table 2.

Area Under the Curve Calculations

	Controls	Group 1 Lean	Group 2 Obese
Number	10	10	10
AUC insulin	4643.3 (1549.02)	3384.62 (465.68)	5012.22 (1467.06)
AUC C-peptide	394.36 (66.84)	421.15 (109.74)	464.92 (112.9)
AUC GIP	101.36 (25.3)^*	49.04 (27.68)	11.75 (12.37)^*
AUC GLP-1	18.95 (11.57)	−1.6 (7.96)	7.18 (3.2)

Data are presented as mean values (standard deviations).

Statistical significance between controls and obese women with PCOS at 0.025 (Bonferroni correction).

AUC, area under the curve.

The IE was calculated from both total insulin and C-peptide secretion. Although analysis failed to detect any significant difference in IE when calculated on the basis of insulin—Kruskal-Wallis, p=0.492, control group: 73.9 (13.4); group 1: 83.72 (43.5); group 2: 79.39 (21.25) (μIU/mL/min)—there was statistical significance when C-peptide was used for calculations—control group: 46.38 (10.24); group 1: 67.86 (12.8); group 2: 88.96 (10.95) (ng mL/min)]. IE was found to be significantly augmented in the obese women with PCOS compared to controls (Student's t test, p=0.011) and this finding remained robust in the subgroup analysis including only BMI-matched healthy women (p=0.015).

C-peptide measurements in the course of the OGTT are shown in Figure 2. C-Peptide levels did not differ between groups (Greenhouse-Geisser correction, p=0.203) yet changed significantly in the course of time in all subjects, as expected (Greenhouse-Geisser correction, p<0.001). However, no difference in the pattern of this change was detected across groups (Greenhouse-Geisser correction, p=0.353).

FIG. 2.

C-peptide levels (ng/mL) in the course of OGTT in control women and lean and overweight/obese women with PCOS. Df, degrees of freedom;¹Greenhouse-Geisser correction;²statistically significant effect.

Discussion

We have studied the incretin response to OGTT and the IE, assessed as the plasma insulin and C-peptide response gradient during an isoglycemic protocol, in a mixed population of lean and obese healthy women and PCOS patients. We chose women with similar insulin sensitivity (Table 1) to avoid a possible compensating effect on beta-cell activity and release. Women with PCOS exhibited a nonsignificant increase in insulin and C-peptide levels during the 3-hour OGTT compared to the control group despite similar glucose levels between the groups. This is in accordance with previous studies that demonstrated increased insulin and C-peptide levels in PCOS independent of insulin sensitivity and obesity.^24
–26

Vrbikova et al.²⁰ demonstrated increased total GIP levels and lower late-phase active GLP-1 concentrations during OGTT in lean women with PCOS compared to BMI-matched and age-matched control subjects. These researchers suggested that the increased GIP levels in PCOS could be attributed to a primary overactivity of the enteroinsular axis and could subsequently lead to an exaggerated stimulation of the beta-cells to secrete more insulin. In another study, which included both lean and obese women with PCOS, incretin hormone response did not differ significantly between healthy and PCOS subjects.²¹ When researchers conducted a subgroup analysis, however, they demonstrated lower GIP levels in obese women with PCOS compared with obese healthy women and lean women with PCOS, suggesting a potential synergistic role of obesity and PCOS in the alteration of GIP response. In our study, overweight and obese women with PCOS also showed a significantly lower GIP response to glucose load compared to BMI-matched controls. We noticed no statistical difference between the lean and obese women with PCOS, however; thus, our results cannot fully support a synergistic role of obesity and PCOS in the alteration of GIP response observed in this study population.

Although both incretins act in an additive manner and regulation of their secretion shares many common features, we failed to detect any significant difference in GLP-1 response after OGTT between the two groups. However, significantly lower baseline GLP-1 levels were observed in obese women with PCOS compared to the controls and the lean women with PCOS. Subgroup analyses with BMI-matched controls did not confirm this significant difference in GLP-1 baseline values. We hypothesize that obesity mostly likely contributed to these results, as it has been reported previously in obese children and young adults.²⁷

The IE for C-peptide was significantly augmented in the overweight/obese women with PCOS compared to the BMI-matched control subgroup. Calculated on the basis of insulin secretion, the IE did not show any significant difference between groups. It has been reported in the literature, however, that insulin secretion could be more reliably estimated from peripheral C-peptide rather than insulin response²⁸ because of a nonconstant insulin extraction from plasma.^29
–31 On the other hand, C-peptide elimination from plasma does not seem to be affected by the level of glycemia,³¹ C-peptide concentration,³² or other factors released by glucose ingestion.³³ This could explain why the numerical values for the apparent IE depend on whether they are calculated from insulin or C-peptide responses, although both polypeptides are secreted into the portal circulation in equimolar amounts.^34,35

The observed increased IE along with the blunted GIP response to OGTT in overweight and obese women with PCOS with normal insulin sensitivity was largely unexpected, as in diabetes, which is the best-studied condition known to affect the enteroinsular axis, the IE is decreased, whereas there is an exaggerated GIP response after oral glucose.¹⁵ On the other hand, older studies had demonstrated an increased activity of the enteroinsular axis in obesity and in peptic ulcer diseases, in terms of hyperinsulinemia and exaggerated response of GIP after meals.³⁶ In an attempt to explain this largely unexpected combination of findings, several methodologic and pathophysiologic interpretations may be supported. A plausible explanation could be based on the limited number of participants, which could have led to underpowered analysis or results vulnerable to the distorting effect of outliers. Furthermore, technical considerations regarding incretin measurements may partially explain the observed physiologic discrepancy. Given that data regarding the enteropancreatic axis in women with PCOS are not conclusive, however, an alternative explanation of the findings can not be excluded. Thus, an increased insulinotropic effect along with a blunted GIP response to OGTT in overweight and obese women with PCOS with normal insulin sensitivity may be explained by a compensatory increase in pancreatic beta-cell sensitivity and responsiveness to incretin stimulation, which can be either an intrinsic characteristic of the syndrome or a result of the interaction between PCOS and obesity on beta-cells.

Conclusions

This study was conducted to estimate the IE in PCOS, independent of insulin resistance. However, the limited number of participants could have led to underpowered analysis; therefore, our results should be interpreted cautiously. Future studies that will also include administration of GIP during hyperglycemic clamp, along with calculation of the IE, could possibly shed more light on the underlying mechanisms involved in hyperinsulinemia in PCOS.

Footnotes

Disclosure Statement

The authors declare no conflicts of interest.

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