Myo-Inositol Enhances the Inhibitory Effect of Metformin on Gonadotropin Levels in Postmenopausal Women

Objectives: Metformin decreased circulating levels of anterior pituitary hormones and its effect on thyrotropin concentration was found to be stronger in individuals receiving myo-inositol. Phospholipids containing inositols are precursors of second messengers of several hormones, including gonadotropins and insulin. The aim of the current study was to investigate whether the concomitant use of myo-inositol changes the effect of metformin on gonadotropin levels. Design: A prospective observational study. Participants/Materials, Setting, and Methods: This study, conducted at a university-affiliated medical center, included two groups of postmenopausal women with prediabetes, matched for age, FSH and LH levels, and insulin sensitivity: women taking myo-inositol preparations for at least 6 months (group A, n = 23) and women not receiving inositol preparations (group B, n = 23). All participants were treated with metformin (850 mg twice daily) for the following 6 months. At the beginning and at the end of the study, we assessed plasma glucose, insulin, FSH, LH, thyrotropin, prolactin, adrenocorticotropic hormone, insulin-like growth factor-1, estradiol, and glycated hemoglobin. Results: The impact of metformin on glucose levels, the homeostatic model assessment 1 of insulin resistance ratio, and glycated hemoglobin was more pronounced in group A than in group B. Metformin administered with myo-inositol reduced both FSH and LH. No significant changes in gonadotropin levels were observed in women receiving only metformin. The impact on FSH and LH levels correlated with their baseline concentrations and with the degree of improvement in insulin sensitivity. Levels of the remaining hormones did not change throughout the study. Limitations: The most important limitation of the study is a relatively small number of participants. Moreover, the study protocol does not allow to conclude whether similar effects are observed in premenopausal women. Conclusions: Myo-inositol may enhance the inhibitory effect of metformin on gonadotropin production in postmenopausal women.

© 2022 The Author(s). Published by S. Karger AG, Basel

Introduction

Recent animal and human studies have shown that metformin inhibits secretory function of overactive gonadotrophs [1-6]. In rats, metformin reduced gonadotropin secretion induced by both gonadotropin-releasing hormone and activin [1]. Administered to women with polycystic ovary syndrome, the drug decreased luteinizing hormone (LH) and the LH/follicle-stimulating hormone (FSH) ratio [2, 3]. The decrease in LH secretion contributed to a reduction in androgen production in metformin-treated individuals with this disorder [7]. In postmenopausal women, high-dose (3 g) metformin treatment reduced FSH concentration [4]. This effect was, however, not observed in postmenopausal women with elevated prolactin levels, receiving hormone replacement therapy [5]. Finally, in hypogonadal men, but not eugonadal men, metformin decreased plasma levels of both FSH and LH [6]. These observations are in line with the previous findings, indicating that metformin reduces elevated levels of thyrotropin and prolactin and that its effect correlates with baseline production of these hormones [8-11]. It seems that the impact on secretory function of anterior pituitary cells results from the fact that the pituitary gland accumulates the largest amounts of metformin among all brain structures [12].

Myo-inositol is the most widely distributed form of inositol, playing a significant role in glucose metabolism and hormone signaling [13]. Phospholipids containing myo-inositol are precursors of second messengers of several hormones, including gonadotropins, thyrotropin, and insulin [14]. Some studies indicate that myo-inositol affects hormone levels. Administered to women with polycystic ovary syndrome together with folic acid, myo-inositol decreased concentrations of LH, prolactin, and testosterone, and the LH/FSH ratio [15]. The impact of myo-inositol/selenium combination therapy on concentration of thyrotropin, free thyroxine, and free tri-iodothyronine was stronger than that of selenium alone [16, 17]. Finally, myo-inositol potentiated the effect of metformin on thyrotropin levels in women with polycystic ovary syndrome and mild thyroid hypofunction [18].

However, the interaction between metformin and myo-inositol at the level of pituitary gonadotrophs has not been understood. To fill in this gap, the current study was aimed at investigating whether concomitant use of myo-inositol determines the impact of metformin on gonadotropin levels.

Materials and Methods

The study protocol adhered to the principles of the Declaration of Helsinki and was approved by the local Ethics Committee. Written informed consent was obtained from all participants before the study began. The paper was prepared in accordance with the Enhancing the Quality and Transparency of Health Research (EQUATOR) Network guidelines for observational studies (STROBE).

Patients

The study population was recruited among postmenopausal women (aged 50–70 years) with prediabetes. To be considered for enrollment, they had to meet all the following criteria of postmenopause: (1) the absence of menstrual periods for at least 12 months, (2) FSH levels above 30 IU/L, and (3) estradiol levels below 110 pmol/L. Prediabetes was diagnosed if any of the following criteria were met: fasting glucose between 100 and 125 mg/dL, 2-h postprandial glucose between 140 and 199 mg/dL or glycated hemoglobin between 5.7 and 6.4%. Only women complying with the lifestyle modification for more than 3 months were included.

The study population included 2 groups of patients: women taking myo-inositol preparations (2 g per day) for at least 6 months before enrollment (group A; n = 23) and women not receiving inositol preparations (group B; n = 23). In order to obtain two groups matched for age, insulin sensitivity, and gonadotropin levels, group B was selected among 60 women, meeting the inclusion and exclusion criteria. One-to-one matching was performed using the propensity score method, and this procedure was carried out with the help of WINPEPI computer program (version number: 3.26, Brixton Health, Llanidloes, United Kingdom).

The exclusion criteria were as follows: diabetes, pituitary, thyroid or adrenal disorders, renal failure, hepatic failure, anemia, malabsorption syndrome, other serious disorders, ongoing pharmacotherapy (with the exception of myo-inositol in group A), and poor patient compliance. The sample size calculation showed that a sample of 20 patients per study group was required to detect a 20% difference between baseline and follow-up values of the primary endpoint (plasma FSH concentration) with 80% power and a significance level of 5%.

Study Design

Over the entire study period, all women were treated with metformin. After 2 weeks, a starting dose of metformin (850 mg once daily) was titrated to the final dose (850 mg twice daily), administered for the following 6 months. To limit seasonal variations in the outcome measures, similar numbers of patients started treatment between March and April (n = 22) and between October and November (n = 24). Women previously treated with myo-inositol (group A) received the same daily dose of this agent as before enrollment. All participants were also instructed to maintain their lifestyle habits during the course of the study. Medication adherence was measured by counting the number of residual tablets.

Laboratory Assays

All measurements were carried out on the first and the last study day. In group A, glucose homeostasis markers, gonadotropins, and estradiol were also assessed before implementation of myo-inositol treatment. Blood samples were collected in the morning (between 8 and 9:00 a.m.) after a 12-h overnight fast and assayed in duplicate. Plasma levels of glucose and whole blood levels of glycated hemoglobin were assessed by an automated system (COBAS Integra 400 Plus, Roche Diagnostics, Basel, Switzerland). Plasma levels of insulin, FSH, LH, thyrotropin, prolactin, estradiol, and anti-Müllerian hormone (only in samples of 11 patients in each group) were assayed by direct chemiluminescence using acridinium ester technology (ADVIA Centaur XP Immunoassay System, Siemens Healthcare Diagnostics, Munich, Germany). Circulating levels of adrenocorticotropic hormone (ACTH) and insulin-like growth factor-1 were measured by solid-phase enzyme-labelled chemiluminescent immunometric assays (Immulite, Siemens, Munich, Germany). The intra- and inter-assay coefficients of variation ranged from 2.5 to 6.0% and from 4.1 to 8.4%, respectively. The minimum detectable concentrations were as follows: 0.6 mIU/L for insulin, 0.3 IU/L for FSH, 0.07 IU/L for LH, 0.01 mIU/L for thyrotropin, 0.3 ng/mL for prolactin, 9 pg/mL for ACTH, 20 ng/mL for insulin-like growth factor-1, 23 pmol/L for estradiol, and 0.30 pmol/L for anti-Müllerian hormone. The homeostasis model assessment of insulin resistance index (HOMA-IR) was calculated as follows: fasting glucose (mg/dL) × fasting insulin (μIU/mL)/405.

Statistical Analysis

All variables were logarithmically transformed to correct for skew. Between-group comparisons were performed by two-sample t tests, while and within-group comparisons using Student’s paired t tests, respectively. The χ2 test was employed to compare nominal data. Correlations between the investigated variables were assessed using Pearson’s r tests for two continuous variables; Phi coefficient for one continuous and one categorical variable; and point-biserial for two categorical variables. Statistical significance was assumed at p < 0.05.

Results

There were no significant differences in age, smoking, body mass index, blood pressure, glucose, HOMA-IR, glycated hemoglobin, and plasma levels of the assessed hormones between women receiving metformin and myo-inositol and women treated with metformin alone (Tables 1, 2). No significant adverse effects were reported over the entire study period and no patient dropped out of the study. Over the entire study period, all participants complied with treatment and dietary recommendations. Cumulative doses of myo-inositol in group A at baseline and follow-up were 640 ± 132 g and 1,004 ± 128 g, respectively. A post hoc power calculation based on the primary outcome data and the given sample size showed that the study had sufficient statistical power (0.85). In group A, glucose, HOMA1-IR, and glycated hemoglobin (but not gonadotropin and estradiol levels) were lower at baseline than before implementation of myo-inositol treatment (online suppl. Table 1; for all online suppl. Material, see www.karger.com/doi/10.1159/000527365.

Table 1.

Baseline characteristics of participants

/WebMaterial/ShowPic/1476988Table 2.

The effect of metformin on glucose homeostasis markers and hormone levels in postmenopausal women with prediabetes

/WebMaterial/ShowPic/1476986

In both groups, metformin reduced plasma glucose, HOMA-IR, and glycated hemoglobin, but this effect was stronger in group A than group B. In group A, the treatment reduced levels of both FSH and LH. No significant changes in gonadotropin levels were observed in group B. Levels of thyrotropin, prolactin, ACTH, insulin-like growth factor-1, estradiol, and anti-Müllerian hormone did not change throughout the study. The study groups differed in follow-up values of glucose, HOMA-IR, glycated hemoglobin, FSH, and LH (Table 2). The body mass index and blood pressure did not change throughout the study period (data not shown).

Treatment-induced changes in FSH levels in group A correlated with its baseline concentrations (r = 0.47; p = 0.002), baseline HOMA-IR (r = 0.37; p = 0.017), and with treatment-induced changes in HOMA-IR (r = 0.46). In group A, there were also correlations between changes in LH levels and baseline LH (r = 0.38; p = 0.012), baseline HOMA-IR (r = 0.32; p = 0.037), and the impact of treatment on HOMA-IR (r = 0.34; p = 0.024). The remaining correlations did not reach the level of significance.

Discussion

In line with our previous report [4], metformin administered to patients not receiving inositol preparations had no effect on FSH and LH levels. This finding, contrasting with FSH-lowering effect of high-dose (3 g) metformin treatment [4], indicates that the impact of this agent on gonadotroph secretory function is dose-dependent. Interestingly, a similar relationship between metformin dose and the strength of action was observed in the case of prolactin [19]. These findings suggest that the pituitary gland, although located outside the blood-brain barrier [20], is characterized by relatively low sensitivity to metformin. Therefore, concentrations of metformin improving glucose homeostasis may be the subthreshold to influence pituitary hormone secretion. Theoretically, we cannot exclude that the impact of low-dose metformin treatment would be significant in patients with higher baseline levels of gonadotropins than in our population.

The novel finding of the current study is that myo-inositol potentiated the impact of metformin on secretory function of human gonadotrophs. Although the same doses (2 g daily) of myo-inositol were found to reduce circulating levels of anterior pituitary hormones in women with polycystic ovary syndrome [15], we did not observe differences in gonadotropin levels during the treatment with only myo-inositol. Because treatment-induced changes in FSH and LH levels did not correlate with the length of myo-inositol treatment and with cumulative doses of myo-inositol, the obtained results cannot be explained by a time-dependent effect of myo-inositol. Between-group differences in metformin action cannot be also attributed to differences in baseline group characteristics because both study arms were well matched for age, HOMA-IR, and gonadotropin levels. Therefore, superiority of metformin/myo-inositol combination therapy to myo-inositol alone suggests the interaction of both agents at the level of gonadotropin-secreting cells.

The impact of metformin/myo-inositol combination therapy on the hypothalamic-pituitary-gonadal axis activity in postmenopausal women was limited to its action on gonadotropin secretion. The treatment had no effect on concentrations of either estradiol or anti-Müllerian hormone, the latter of which is produced by granulosa cells of small, growing follicles in the ovary and is regarded as a marker of ovarian reserve [21]. Very low follow-up values of both hormones indicate that metformin, even if administered together with myo-inositol, does not have any influence on ovarian function in postmenopausal women.

We can only speculate about the clinical significance of our findings. Because the reduction in FSH and LH levels correlated with baseline gonadotropin concentrations, the impact of metformin/myo-inositol combination therapy on gonadotroph secretory function is greatest in individuals with the highest gonadotropin concentrations. This suggests that the combination therapy may be useful in postmenopausal women with functioning gonadotroph adenoma or with ectopic gonadotropin secretion if surgical resection of the tumor is not successful, patients are not eligible for surgery or refuse surgical intervention. Because age-related elevation in LH concentration impairs cognitive functions [22], metformin/myo-inositol combination therapy may have some protective effect against neurodegenerative disorders. Finally, because FSH was found to stimulate osteoclastogenesis in an estrogen-independent manner [23], the decrease in gonadotropin production may slow down the development and progression of postmenopausal osteoporosis.

Another important observation of the current study is that metformin/myo-inositol combination therapy was superior to metformin alone in affecting glucose homeostasis in subjects with prediabetes. The beneficial effect of myo-inositol alone was reported previously in both animals and humans and may be explained by its modulatory action on the glycosyl phosphatidylinositol/inositol phosphate glycan signaling pathway, acting as a messenger of insulin action [24-27]. Previous studies comparing metabolic effects of both studied agents included only or mainly women with polycystic ovary syndrome, showing that the impact of myo-inositol on glucose homeostasis was either similar [28] or even more pronounced [29] than that of metformin. Interestingly, metformin administered to women with polycystic ovary syndrome together with myo-inositol was superior to metformin alone in reducing HOMA-IR [30], although differences between both arms were less pronounced than in the present study. The obtained results indicate that insulin-resistant postmenopausal women may be another group of patients benefiting from metformin/myo-inositol combination therapy. Although in the current study myo-inositol supplementation was accompanied by a reduction in levels of glucose, HOMA1-IR, and glycated hemoglobin, add-on metformin therapy produced further improvement in glucose homeostasis markers. No differences in glucose homeostasis markers in women refusing metformin treatment after 6 and 12 months of myo-inositol administration [Krysiak et al., unpubl. observations] suggest that our findings reflect interactions between metformin and myo-inositol.

The exact molecular mechanism responsible for the gonadotropin-lowering effect of metformin/myo-inositol combination therapy is unknown. This effect cannot be explained by the impact on FSH signaling because postmenopausal ovaries are resistant to gonadotropin stimulation. The presence of correlations with both baseline and treatment-induced changes in HOMA-IR suggests its association with the treatment-induced improvement in insulin sensitivity. The most likely explanation is its effect on activity of adenosine 5′-monophosphate-activated protein kinase (AMPK), which is a nutrient and energy sensor that maintains energy homeostasis and is essential to maintain glucose balance [31]. AMPK plays an important role in mediating many metabolic effects of metformin and the drug affects AMPK activity also in the brain [32]. Gonadotrophs are characterized by higher expression of AMPK than most other pituitary cells and AMPK mediates the inhibitory effect of metformin on gonadotropin secretion in animals [1]. Because inositol polyphosphate multikinase downregulates AMPK activity [33], treatment with its precursor (myo-inositol) may potentiate the inhibitory effect on the AMPK signaling pathway. It is also possible that metformin interacts with myo-inositol at the level of the tuberoinfundibular dopamine pathway. In line with this explanation, activation of this pathway inhibits gonadotropin release [34], while metformin was found to stimulate dopaminergic transmission in the central nervous system [35]. Moreover, deficiency of phosphatidylinositol (3,4,5)-trisphosphate, which is a second messenger in the insulin pathway [13], impairs dopaminergic transmission in the brain [36] and it is possible that myo-inositol administration, by increasing phosphatidylinositol (3,4,5)-trisphosphate content, may, in concert with metformin, stimulate central dopaminergic transmission.

Apart from a stronger effect on gonadotropin levels and insulin sensitivity, metformin/myo-inositol combination therapy may be associated with other benefits for postmenopausal women. Both metformin and myo-inositol improved or prevented deterioration of endothelial dysfunction, which is a key player in the initiation and progression of cardiovascular disease [37, 38]. Although soy isoflavones [39] and other prenylflavonoids [40] were found to exert a beneficial effect on endothelial function in healthy postmenopausal women, metformin and myo-inositol may be an alternative of flavonoids in postmenopausal women with impaired insulin sensitivity. Administration of metformin and myo-inositol combination therapy may be also beneficial from the sexual point of view. Urogenital aging, resulting from estrogen deprivation after ovarian function cessation, is observed in most postmenopausal women. This process may be, however, slowed down by both estradiol and non-estrogenic (oxytocin, dehydroepiandrosterone, or ospemifene) therapies [41, 42]. It is possible that metformin and myo-inositol alleviate symptoms of urogenital aging in insulin-resistant postmenopausal women. In line with this suggestion, the prevalence of sexual dysfunction is greater in insulin-resistant postmenopausal women than in healthy postmenopausal women [43]. Moreover, metformin was found to improve all aspects of female sexual functioning in premenopausal women with diabetes and prediabetes, and this effect correlated with the degree of improvement in insulin sensitivity [44].

The current study has several strengths. To the best of our knowledge, our research is the first study comparing the impact of metformin/myo-inositol combination therapy and metformin monotherapy on pituitary hormone levels. Owing to the strict inclusion and exclusion criteria, the studied population was a relatively homogeneous group of patients. Beyond the hypothalamic-pituitary-ovarian axis activity, the study assessed secretory function of the remaining types of anterior pituitary cells. Finally, the obtained results seem to be important from a practical point of view because insulin resistance and its complications are common health problems in postmenopausal women and often require treatment with insulin-sensitizers.

As with the majority of studies, the design of the current study is subject to limitations. A small number of participants (although the study had sufficient statistical power), as well as relatively short treatment duration make drawing any strong conclusions difficult. Because of the inclusion criteria, it is not certain whether similar effects of metformin and myo-inositol are observed in postmenopausal women with diabetes or normal glucose tolerance. It cannot be also ruled out that the effect of treatment on gonadotropin levels is different in premenopausal women and men with hypogonadism. Because the study protocol did not provide a direct experimental or mechanistic link between the effect of both studied agents and gonadotropin levels, our conclusions are only speculative. Finally, although we tried to minimize the impact of random diurnal, seasonal, and analytical variations in the investigated variables, we cannot totally exclude the regression toward the mean effect.

Summing up, metformin administered at the daily dose of 1.7 g treatment reduced gonadotropin levels only in myo-inositol treated patients. The impact of this agent on plasma levels of FSH and LH correlated with the treatment-induced improvement in insulin sensitivity. This finding indicates that myo-inositol may enhance the effect of metformin on gonadotroph secretory function in individuals with elevated gonadotropin levels. Because of the pilot nature of the study, these findings need to be confirmed in a larger clinical trial.

Statement of Ethics

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (the Bioethical Committee of the Medical University of Silesia -KNW/0022/KB/208/17; May 16, 2017). All participants provided written informed consent prior to enrolment in the study.

Conflict of Interest Statement

The authors have no conflicts of interest to disclose.

Funding Sources

The study was supported by the statutory grant of the Medical University of Silesia (PCN-2-072/N/1/Z).

Author Contributions

Robert Krysiak conceived the study, participated in its design, performed the statistical analysis, as well as drafted and edited the manuscript. Karolina Kowalcze conducted the literature search, carried out the assays, and performed the statistical analysis. Bogusław Okopień participated in the study design and coordination, and provided critical input during manuscript preparations. All the authors read and approved the final manuscript.

Data Availability Statement

All data generated or analyzed during this study are included in this manuscript. Further inquiries can be directed to the corresponding author.

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