Investigating the potential role of swertiamarin on insulin resistant and non-insulin resistant granulosa cells of poly cystic ovarian syndrome patients

Polycystic ovarian syndrome (PCOS) is a multifaceted disease and an approach to decrease this has become a top priority for many health organizations [1]. The life style factors such as physical exercise, psychological stress, high carbohydrate diet and sedentary life that impact fertility are modifiable and are considered to be the first-line of treatment in PCOS [2‐4]. Symptom oriented pharmacological intervention such as oral contraceptive to regulate menstrual cycles and decrease hirsutism, spironolactone that block androgen receptors, finasteride to block production of active form of testosterone and insulin sensitizers such as metformin, thiazilidinediones (TZD’s) and D-chiro Inositol are in use for improving insulin sensitivity and lipid levels accompanied by weight loss [1, 5, 6]. Psychosocial interventions have also proven to improve PCOS-related mental health issues [7].

Considering the role of insulin resistance (IR) in the interplay of metabolic and reproductive aberrations in infertility, insulin sensitizing drugs (ISD) are expected to have beneficial effects by restoring ovulatory menstrual cycles, reducing insulin resistance and thus being important therapeutic modality for PCOS [8, 9]. Insulin sensitizers such as the thiazolidinediones (TZD’s), D-chiro-Inositol, and metformin are postulated to improve insulin sensitivity and several other aspects of the syndrome, including reproductive abnormalities [10, 11]. TZD’s improve insulin sensitivity in PCOS through decrease in hepatic glucose production [11]. Studies with TZD’s have reported fetal growth restriction as a potential risk in animal experiments and high incidence of weight gain among the users that further hampers their use in obese women with PCOS [12, 13]. D-chiro-Inositol has been observed to show effect on serum hormone binding globulin levels without affecting testosterone, fasting glucose, fasting insulin, and ovulation rate [14]. Supplementation of insulin sensitizer myo-inositol has demonstrated positive effects in oocyte quality of PCOS patients and post-menopausal transitions [15‐17].

However, it’s role in improving clinical pregnancy rates is under controversy and is not recommended for normo insulinemic lean or obese PCOS [11, 18, 19]. Thus, none of the ISD’s could increase a chance of having a live birth and are limited by the prevalence of contraindications in women with PCOS [1, 10, 20]. Moreover with limited understanding of their mechanism, the type of patients, whether only PCOS-IR or even PCOS-NIR would be benefitted by their effects remains to be identified [21].

A number of herbal medicines such as Vitex agnus-castus, Cimicifuga racemosa, Tribulus terrestris, Glycyrrhiza spp*., Paeonia lactiflora, Cinnamomum cassia and Aloe vera can improve ovarian function, androgen excess, obesity, insulin resistance, blood lipids and inflammation and exert beneficial effects in PCOS [1, 6, 22‐24]. Enicostemma Littorale blume is a herbal plant that is known for its anti-diabetic effect but its bio-active molecules has been unexplored in the field of PCOS.

Enicostemma Littorale blume (EL) has been widely used by folks since ages for the treatment of diabetes. During last ten years our lab worked on EL and unravelled its hypo gycemic, hypo lipidemic, anti-inflammatory and insulin sensitizing potentials in different animal models [25‐28]. Swertiamarin, being its principal compound has proved to be a potent insulin sensitizer in STZ-NA diabetic rat models and different cell lines [29, 30]. The present study focuses to investigate direct effect of bio-active molecule swertiamarin on reproductive endocrinology for the treatment of women with PCOS.

Certain obese and lean women with PCOS are observed to have normal insulin sensitivity, hence it would be interesting to discover potential bio actives for treating PCOS-IR and NIR, considering the side effects observed by conventional pharmaceutical drugs. [31]. PCOS patients were classified as IR and NIR based on a novel molecular marker insulin receptor – β (INSR-β) expression on luteinized granulosa cell as shown in our previous paper [32]. The effects of swertiamarin and metformin have been explored on granulosa cell death, insulin and steroidogenic signaling, genes involved in fatty acid metabolism and steroidogenic hormones in granulosa cells from PCOS-IR and PCOS-NIR.

The study was approved by the Institutional Ethics committee for human research (IECHR), Faculty of Science, The M. S. University of Baroda, Vadodara (Ethical Approval Number FS/IECHR/BC/SG2).

Ever since the role of IR in the pathogenesis of PCOS has been established, a positive effect of insulin-sensitizing drugs in the treatment of PCOS has been demonstrated. In accordance with this, our study is the first one to report for the potential of swertiamarin, a bio active herbal insulin sensitizers for amelioration of IR and reestablishment of steroidogenesis in hLGC’s isolated from follicular fluid of PCOS-IR and PCOS-NIR patients using metformin as a positive control.

Insulin resistance in PCOS has been associated with increase in granulosa cell death [36]. The recovery of hLGC viability after incubation with swertiamarin in our study may be interpreted as indicative of reduced susceptibility of the PCOS-IR cells to undergo apoptosis as is observed with metformin in earlier studies [34]. However, swertiamarin did not show any effect on cell viability in hLGC from PCOS-NIR indicating the process of granulosa cell death in PCOS-NIR to be other than IR.

It is well known that ISD’s can modulate insulin signalling that can recruit its downstream docking proteins to activate several other signalling pathways in different cell types [18, 37]. Swertiamarin is well known for its anti-diabetic and anti-hyperlipidemic effects in various animal models, different cell lines and clinical trials with humans. Our results for the first time show a direct interaction of swertiamarin with key components of the classical insulin-signaling pathway thereby highlighting their ability to sensitize IR condition in hLGC’s from PCOS-IR. Moreover swertiamarin with a dose of 66 μM seemed to be a potent insulin sensitizing drug for reversing insulin sensitivity in PCOS-IR as compared to metformin 1 mM. Surprisingly metformin increased the basal expression of key signalling proteins in the insulin signalling pathway which might result in increased insulin sensitivity and decreased insulin levels to less than normal even in PCOS-NIR group in the long term.

The importance of P38 MAPK and ERK1/2 in PCOS granulosa cells has been demonstrated in studies indicating their over expression as a result of oxidative stress and inflammation leading to decrease in the expression of StAR and progesterone synthesis. [38‐41]. Decrease in the protein expression of pP38 and ERK1/2 MAPK in treated hLGC’s from PCOS-IR as well as PCOS-NIR indicating reversal of oxidative stress and thus reversal of cell death. Our findings are in line with the literature where swertiamarin could enhance anti-oxidant defense system by suppressing oxidative stress and attenuate inflammation and apoptosis [30, 42].

Lipogenic genes being the regulators of the accumulation of lipids instead of cholesterol in granulosa cells, their profile was assessed. As insulin signalling is related to lipid metabolism and lipids are important for oocyte maintenance, we further observed the effect of swertiamarin on the lipogenic genes SREBP1c along with enzymes ACC-1, FAS and CPT-1 in PCOS-IR and PCOS-NIR condition. Our data indicated that SREBP-1c protein expression, in addition to the expression of lipogenic target genes ACC1 and FAS and fatty acid oxidation gene CPT-1 were suppressed by swertiamarin in hLGC’s from PCOS-IR supported by [30, 43]. The reversal of these processes explain the association of swertiamarin supplementations in decreasing lipid accumulation in granulosa cells seemed with an improvement in granulosa cell metabolism via the regulation of SREBP-1c, ACC-1, FAS and CPT-1 expression.

We further wanted to study the effect of the bio active on IGF (IGF-I, IGF-II, IGF-1R and IGF-2R) system whose involvement is appreciated in the development of preantral to preovulatory follicles, in the process of follicular atresia and is over expressed in diabetic condition [44‐47]. In the present chapter, treatment of swertiamarin reversed the aberrant effects of PCOS on IGF-I, IGF-II, IGF-1R and IGF-2R in hLGC’s from PCOS-IR. Studies in literature with administration of metformin in diabetic patients have reported upregulation of IGF-1 gene and down regulation of its receptor thereby improving insulin sensitivity and glucose uptake [48‐52].

As observed with the proteins involved in insulin signalling system, metformin reversed the expression of IGF-2R in hLGC’s from PCOS-NIR. These findings combined with no change in basal expression of these proteins and genes in control hLGC’s as well as PCOS-NIR hLGC’s strongly suggest that other than metformin, swertiamarin might function only during IR condition.

The process of steroidogenesis is very crucial for the development of oocyte, its fertilization and embryo implantation [53]. In PCOS condition irrespective of IR, gene and protein expression of steroidogenic factors and their corresponding steroid hormones are lowered [8, 53]. In the present study swertiamarin could revert back mRNA expression of gonadotropin receptors, mRNA and protein expression of StAR, CYP11A1, CYP19A1, 17β-HSD and 3β-HSD, their enzyme activity along with secretion of the corresponding steroid hormones estradiol and progesterone in hLGC’s from PCOS-IR thus improving the process of steroidogenesis. Metformin, the positive control of the study improves steroidogenesis by down regulating FSH-R and increasing the progesterone secretion by hLGC from PCOS women [8, 21]. Swertiamarin at a concentration of 66 μM proved to be a better drug in reversing steroidogenesis with the same potency as compared to metformin 1 mM in PCOS-IR. In the present study testosterone could not be detected in the cell culture supernatant. The finding was consistent with the literature explaining presence of the enzyme P450c17/CYp17, responsible for converting C21 steroids (progestrogens) to C19 steroids (androgens) solely in theca cells and not in granulosa cells [54].

An additional aim of this study was to determine whether swertiamarin and metformin could ameliorate decreased steroidogenesis in hLGC’s from PCOS-NIR. Strikingly, swertiamarin did not ameliorate steroidogenesis. On the basis of the findings that swertiamarin restored insulin sensitivity in PCOS-IR with no effect on PCOS-NIR, it is quite possible to speculate that swertiamarin could be mediating their effects on granulosa cell steroidogenesis only through insulin signaling. Metformin reversed back the decrease in steroidogenesis although with a less pronounced effect as compared to PCOS-IR. This effect could be attributed to the direct effect of metformin on steroidogenesis probably through a multipathway reaction with ERK1/2, pP38 MAPK or PI(3)K all involved in regulation of steroidogenesis [55‐57]. Such direct effects of metformin have been supported by some clinical studies in which the insulin sensitizer increased the ovulation rate, and fertilization rates by having no effect on the basal insulin levels [13, 31, 58]. These findings indicate the possibility of some other factors in hLGC’s from PCOS-NIR yet unidentified and not associated with insulin signalling to have a role in restoring steroidogenesis by metformin. However few studies with metformin in clinical trials have reported increased ovulation rates with decreased basal insulin levels in normo insulinemic subjects indicating hypoglycaemia if prescribed to PCOS-NIR [31, 59]. Moreover as the actual treatment time for metformin to induce a clinical ovulation is 6 months, other drugs are preferred over metformin for rapidly inducing ovulation in PCOS [60]. D-chiro Inositol, an insulin sensitizer, has been observed to induce ovulation in non insulin resistant PCOS via modulating aromatase expression [61].

Collectively the results support the notion that, swertiamarin at 66 μM show effect as insulin sensitizers for alleviating IR condition. Swertiamarin had no effect on steroidogenesis in PCOS-NIR. Metformin did restore steroidogenesis in PCOS-NIR but it is important however to highlight that it decreases the basal insulin signalling parameters in PCOS-NIR group which might lead to adverse effects in the long-term health (Fig. 6). This calls for a proper diagnosis of IR condition in PCOS so that targeted therapy can be prescribed to achieve increased pregnancy rates with decreased time.