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. 2019 Aug 12;18(19):2538–2549. doi: 10.1080/15384101.2019.1652036

Metformin and sitagliptin combination therapy ameliorates polycystic ovary syndrome with insulin resistance through upregulation of lncRNA H19

Qiong Wang a,✉, Jing Shang a, Yun Zhang a, Wei Zhou b,✉
PMCID: PMC6738908  PMID: 31405334

ABSTRACT

Insulin resistance (IR) is prevalent in women with polycystic ovary syndrome (PCOS). Improvement in insulin sensitivity remains one of the most effective treatment strategies for women with PCOS. This study aims to investigate the efficacy and potential mechanism of the combination therapy with metformin (DMBG) and sitagliptin (TECOS) in PCOS. To address this, insulin was used to treat rat ovarian granulosa cells to establish the cellular PCOS model. Insulin and human chorionic gonadotropin (HCG) were subcutaneously injected into SD rats to establish a rat model of hyperandrogenism with pathogenesis similar to PCOS. Our results showed that co-treatment with TECOS and DMBG attenuated the induced apoptosis and insulin resistance (IR) in PCOS model cells, and improved reproductive hormone disorders, ovarian polycystic changes, and IR of PCOS rats. Mechanistically, upregulation of H19 by H19-expressing lentiviruses enhanced efficacy of combination therapy. Furthermore, co-treatment with TECOS and DMBG induced H19 expression via suppressing the PI3K/AKT-DNMT1 pathway. Collectively, these findings demonstrate that combination treatment with TECOS and DMBG ameliorates PCOS with IR, at least partially, through upregulation of lncRNA H19.

KEYWORDS: Polycystic ovary syndrome, metformin, sitagliptin, H19, PI3K/AKT

Introduction

Polycystic ovary syndrome (PCOS) is a common endocrine condition that affects 5–15% of women in reproductive age. The diagnostic criteria include two out of three features: hyperandrogenism, polycystic ovary (PCO) morphology on ultrasound and menstrual irregularities (Rotterdam Criteria 2003) [1,2]. Insulin resistance (IR) is prevalent in PCOS women and is critically involved in reproductive and metabolic complications of PCOS [3,4]. As a compensatory response to IR, hyperinsulinemia develops in this context and disrupts ovarian function, with enhanced androgen production and arrest of ovarian follicular development [4]. Therefore, improvement in insulin sensitivity remains one of the most effective treatment strategies for women with PCOS.

Insulin-sensitizing agents such as metformin (DBMG) may be effective in treating PCOS-related anovulation. Notably, a combination therapy demonstrates greater efficacy than its individual components [5]. Sitagliptin (TECOS) was the first DPP-4 inhibitor approved by the FDA for the treatment of type 2 diabetes. It has been previously observed that TECOS in adjunct to DMBG prevented weight regain more effectively than DMBG alone in obese women with PCOS previously treated with liraglutide [6]. However, the efficacy and potential mechanism of the combination therapy with DMBG and TECOS in PCOS remain unclear.

A significant difference in DNA methylation levels has been observed between PCOS and normal ovaries, suggesting that DNA methylation is associated with the pathogenesis of PCOS [7]. Long non-coding RNAs (lncRNAs) are defined as transcripts longer than 200 nucleotides without coding potential and play critical roles in the pathogenesis of various human diseases, including PCOS [8,9]. Evidence indicates that expression of lncRNAs can be regulated by DNA methylation. For example, overexpression of DNA methyltransferase 1 (DNMT1) leads to hypermethylation of H19 promoter and subsequent downregulation of H19 [10]. Besides, H19 expression was decreased in ovarian granulosa cells of PCOS patients [8], suggesting that the H19 gene may be methylated in PCOS.

Evidence indicates that activation of the phosphoinositide-3 kinase (PI3K)/AKT signaling pathway leads to phosphorylation of DNMT1, which increases DNMT1 nuclear translocation and activity, leading to methylation of downstream genes [11]. PI3K/AKT signaling pathway is activated in PCOS-IR rats, while metformin inhibits PI3K/AKT signaling pathway [12]. Thus, we hypothesized that combination treatment with TECOS and DMBG may inhibit the PI3K/AKT signaling pathway and subsequently inhibit DNMT1 phosphorylation and nuclear translocation, leading to suppression of H19 methylation and upregulation of H19 expression. In this study, we investigated the efficacy of the combination treatment with TECOS and DMBG in PCOS-IR and explore whether H19 upregulation was involved in this process.

Materials and methods

Isolation and identification of rat ovarian granulosa cells

Rat ovarian granulosa cells were isolated from female SD rats as previously described [13], with some alterations. Briefly, female SD rats (21–25 days old) were subcutaneously treated with 40 IU of pregnant mare serum gonadotropin (PMSG). Forty-eight hours after dosing with PMSG, the animals were euthanized by CO2 asphyxiation, and the ovaries were aseptically removed, washed with PBS and then placed in pre-cooled DMEM/F12 medium. The follicles were punctured with a 25-gauge needle under a dissecting microscope, and the granulosa cells were released into DMEM/F12 medium and filtered through a 200-mesh stainless steel cell strainer. After centrifugation for 8 min at 800 r/min, granulosa cells were diluted with DMEM/F12 medium (containing 15% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin) to a final concentration of 3 × 105 live cells/mL and added to culture dishes. Following 48 h of incubation in humidified air at 37°C with 5% CO2, media was changed every two days.

After 48 h of incubation, the cells were observed under an inverted microscope and photographed. The granulosa cells specifically express the follicle stimulating hormone receptor (FSHR), and the cultured ovarian granulosa cells were identified by FSHR immunofluorescence staining according to conventional procedures.

Enzyme-linked immunosorbent assay (ELISA)

The levels of estradiol (E2), follicle-stimulating hormone (FSH), testosterone (T), luteotropic hormone (LH), and progesterone (P) in rat sera or rat ovarian granulosa cells were measured using their commercially available ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China).

Cell apoptosis assay

The cell apoptosis was analyzed using One Step TUNEL Apoptosis Assay Kit (Red Fluorescence) (Beyotime, Shanghai, China) following the manufacturer’s protocol. The images of the Cyanine 3 -labeled TUNEL-positive cells were captured using a fluorescent microscope (Nikon Corporation). The nick-ends labeled in red indicated the apoptotic cells and the cell nuclei were labeled in blue by DAPI.

Western blot

Proteins were extracted from rat granulosa cells or ovary samples using RIPA buffer (Beyotime). The protein concentrations were determined by BCA assay. Then the protein samples were loaded and separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking the nonspecific binding sites with 5% fat-free milk, the membranes were incubated with the following primary antibodies against IRS-1 (insulin receptor substrate 1; Abcam), GLUT4 (glucose transporter type 4; Abcam), PI3K (Abcam), AKT (Abcam), p-AKT (Abcam), DNMT1 (Santa Cruz Biotechnology), and p-DNMT1 (PLlabs, Canada), followed by incubations with secondary antibodies horseradish peroxidase-conjugated IgG. Quantity One (version 4.6.9) was used to quantify the relative band intensities from western blot images. β-actin served as the loading control.

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from rat granulosa cells or ovary samples using TRIzol reagent (Invitrogen). RNA was reverse transcribed into cDNAs using the iScript cDNA synthesis kit (Bio-Rad, CA). The cDNA template was amplified through qRT-PCR using SYBR Green PCR master mix by the ABI7900 system (Applied Biosystem, USA). The expression of H19 was calculated by the 2−ΔΔCt method and normalized to the internal control GAPDH. The primer sequences were as follows: H19 Forward 5ʹ-GGCGATGAACTGGACAAC-3ʹ, Reverse 5ʹ- CCGAAGTAGGAAAGGAGGC-3ʹ; GAPDH Forward 5ʹ-CAAGTTCAACGGCACAGTCAA-3ʹ, Reverse 5ʹ-CGCCAGTAGACTCCACGACA-3ʹ.

Stable expression of H19

The lentiviral pcDNA3.1-YFP-puro-H19 expression vector (LV-H19) and pcDNA3.1-YFP-puro vector control (LV-NC) were designed and synthesized by GenePharm Co. (Shanghai, China). The LV-H19 and LV-NC were infected into rat ovarian granulosa cells. Stable cells were selected by puromycin (1.0 µg/mL, Sigma) and expression of H19 was examined by the qRT-PCR assay.

Immunofluorescence

Briefly, when the cell confluence reached 75–85%, cells were fixed with 4% paraformaldehyde, permeabilized in PBS containing 0.3% Triton X-100 for 1 min, and blocked with normal goat serum for 30 min. Cells were then incubated with primary anti-DNMT1 (1 µg/mL) overnight at 4°C, followed by the Alex Fluor® 488-labeled secondary antibody (green; 1:1000) at 37°C for 1 h. Cells were then washed three times with PBS and stained with DAPI in the dark. The nuclear translocation of DNMT1 was analyzed under a fluorescence microscope (Nikon Corporation, Tokyo, Japan).

Animal experiments

Female SD rats (6-week-old, 180–200 g) were housed under specific pathogen-free (SPF) conditions at the animal experimental center of Henan Provincial People’s Hospital. The experimental protocols were approved by the Ethics Committee of Henan Provincial People’s Hospital. These rats were randomly divided into five groups (n = 8/group): Normal, PCOS, TECOS+DMBG, TECOS+DMBG+LV-NC, and TECOS+DMBG+LV-H19 group. LV-H19 and LV-NC were injected into rat ovaries 30 d before PCOS modeling. The mice in the normal group received a subcutaneous injection of normal saline. The other four groups received subcutaneous injection of insulin combined with human chorionic gonadotropin (HCG) to establish a rat model of hyperandrogenism with pathogenesis similar to PCOS. From Day 1 to Day 10, the rats were injected with slowly increasing doses of insulin, from 0.5 IU/d to 6.0 IU/d. Between Day 11 to Day 22, the insulin was injected at a dose of 6.0 IU/d concurrently with HCG (6.0 IU/d) injected twice per day. After successful modeling, TECOS (10 mg/kg/d) and DMBG (300 mg/kg/d) were administered intragastrically for another 12 d. Rats were sacrificed 24 h after the end of drug administration.

Biochemical analysis

Insulin resistance was measured according to the homeostasis model assessment for insulin resistance (HOMA-IR). Briefly, the venous blood of the rats was extracted 12 hours after fasting, and the fasting blood glucose (FBG) and fasting insulin (FINS) were examined using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The degree of the HOMA-IR was quantified as HOMA-IR = FBG (mmol/L)-FINS (mU/L)/22.5, and HOMA-IR > 2.8 was considered as insulin resistant. Serum levels of FSH, T, LH, E2, and P were measured using their commercially available ELISA kits.

Histopathological evaluation

Ovaries of the rats were fixed in 10% neutral formalin, and then embedded in paraffin before being cut into 4-μm thickness. The sections were stained with hematoxylin and eosin (HE) following the routine procedures. The histological changes were observed and examined using a light microscope (Nikon E100; Nikon Corp, Japan).

Statistical analysis

All statistical analyses were performed using GraphPad Prism version 7.0. The unpaired Student’s t-test was used to analyze differences between the two groups. One-way analysis of variance (ANOVA) was used to analyze differences among multiple groups. The data are presented as the mean ± standard deviation (SD) from three independent experiments. p < 0.05 was considered to indicate a statistically significant difference.

Results

Identification of rat ovarian granulosa cells

After 48 h of incubation, the isolated rat ovarian granulosa cells were observed to display fusiform or polygonal in shape under the inverted microscope (Figure 1(a)). Granulosa cells are the only cells that can express FSHR in the ovary. FSHR immunofluorescence staining showed that over 95% of the cultured cells were ovarian granulosa cells (FSHR-positive, green, Figure 1(b)).

Figure 1.

Figure 1.

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Identification of granulosa cells.

Rat ovarian granulosa cells were isolated from female SD rats. (a) After 48 h of incubation, the cells were observed to display fusiform or polygonal in shape under the inverted microscope. Scale bar: 200 μm. (b) FSHR immunofluorescence staining showed that over 95% of the cultured cells were ovarian granulosa cells (FSHR-positive, green).

Co-treatment with TECOS and DMBG attenuated the induced apoptosis and IR in PCOS model cells

To investigate the effect of TECOS and DMBG on PCOS, we treated rat ovarian granulosa cells with insulin to mimic a cellular model of PCOS. Co-treatment with TECOS and DMBG significantly decreased the increased levels of E2 and LH (Figure 2(a)) and inhibited the enhanced apoptosis in insulin-treated PCOS cells (Figure 2(b)). Furthermore, co-treatment with TECOS and DMBG restored the decreased levels of IRS-1 and GLUT4 in insulin-stimulated PCOS cells (Figure 2(c)). Decreased activity of IRS-1 and GLUT4 is associated with IR [14]. Therefore, these data indicated that co-treatment with TECOS and DMBG attenuated the insulin-induced granulosa cell apoptosis and IR. The efficacy of co-treatment with TECOS and DMBG was stronger than that of treatment with DMBG alone (Figure 2).

Figure 2.

Figure 2.

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Co-treatment with TECOS and DMBG attenuated the induced apoptosis and IR in PCOS model cells.

Rat ovarian granulosa cells were treated with insulin (1.0 μmol/L) for 48 h to mimic a PCOS cellular model. The PCOS cells were then treated with metformin (DMBG) or/and sitagliptin (TECOS). (a) The secretion of E2 and LH was detected by ELISA. (b) Cell apoptosis was detected by TUNEL staining. The nick-ends labeled in red indicated the apoptotic cells and the cell nuclei were labeled in blue by DAPI. Scale bar: 50 μm. (c) The protein levels of IRS-1 and GLUT4 were examined using western blot. *p < 0.05, **p < 0.01, vs. Control; #p < 0.05, ##p < 0.01, vs. PCOS. The data are presented as the mean ±SD (n = 3).

Upregulation of H19 enhanced the TECOS-DMBG co-treatment-mediated attenuation of apoptosis and IR in PCOS model cells

We also found that co-treatment with TECOS and DMBG restored the decreased H19 expression in PCOS model cells (Figure 3(a)). To further investigate whether co-treatment with TECOS and DMBG attenuated the induced apoptosis and IR in PCOS model cells via upregulating H19, we infected the PCOS cells with LV-19 to overexpress H19, followed by co-treatment with TECOS and DMBG. The results from qRT-PCR confirmed that H19 was successfully overexpressed in cells (Figure 3(b)). Furthermore, compared with the TECOS and DMBG group, H19 overexpression further decreased the levels of E2 and LH (Figure 3(c)), inhibited cell apoptosis (Figure 3(d)), and increased levels of IRS-1 and GLUT4 (Figure 3(e)). These data indicated that upregulation of H19 expression notably enhanced the TECOS-DMBG co-treatment-mediated attenuation of apoptosis and IR.

Figure 3.

Figure 3.

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Upregulation of H19 enhanced the TECOS-DMBG co-treatment-mediated attenuation of apoptosis and IR.

(a) H19 expression in rat ovarian granulosa cells in the four groups was examined using qRT-PCR. (B-E) Rat ovarian granulosa cells were divided into control, PCOS (stimulated with 1.0 μmol/L insulin for 48 h), TECOS (sitagliptin) + DMBG (metformin), TECOS+DMBG+LV-NC, and TECOS+DMBG+LV-H19 group. (b) The overexpression efficiency of H19 in rat ovarian granulosa cells was confirmed by qRT-PCR. (c) The secretion of E2 and LH was detected by ELISA. (d) Cell apoptosis was detected by TUNEL staining. The nick-ends labeled in red indicated the apoptotic cells and the cell nuclei were labeled in blue by DAPI. Scale bar: 50 μm. (e) The protein levels of IRS-1 and GLUT4 were examined using western blot. *p < 0.05, **p < 0.01, vs. Control; #p < 0.05, ##p < 0.01, vs. PCOS; &p < 0.05, &&p < 0.01, vs. TECOS+DMBG+LV-NC. The data are presented as the mean ±SD (n = 3).

Co-treatment with TECOS and DMBG induced H19 expression via suppressing PI3K/AKT-DNMT1 pathway

Evidence indicates that activation of the PI3K/AKT signaling pathway leads to phosphorylation of DNMT1, which increases DNMT1 nuclear translocation and activity, leading to methylation of downstream genes [11]. Besides, DNMT1 overexpression leads to hypermethylation of H19 promoter and subsequently downregulation of H19 expression [10]. Thus, we explored whether TECOS and DMBG co-treatment induced H19 expression via regulating the PI3K/AKT-DNMT1 pathway. The protein levels of PI3K and phosphorylation levels of AKT and DNMT1 were significantly up-regulated in insulin-induced PCOS cells (Figure 4(a)). Furthermore, an increased DNMT1 nuclear translocation was observed in insulin-induced PCOS cells (Figure 4(b)). Importantly, co-treatment with TECOS and DMBG inhibited the insulin-induced PI3K/AKT activation as well as DNMT1 nuclear translocation and activity (Figure 4).

Figure 4.

Figure 4.

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Effect of co-treatment with TECOS and DMBG on levels of PI3K/AKT-DNMT1 pathway-related proteins.

Rat ovarian granulosa cells were divided into control, PCOS (stimulated with 1.0 μmol/L insulin for 48 h), and TECOS (sitagliptin) + DMBG (metformin) group. (a) The protein levels of PI3K, AKT, p-AKT, DNMT1, and p-DNMT1 were examined using western blot. (b) The nuclear translocation of DNMT1 was analyzed under a fluorescence microscope. The green signals indicated DNMT1 and the cell nuclei were labeled in blue by DAPI. Scale bar: 50 μm. **p < 0.01, vs. Control; #p < 0.05, ##p < 0.01, vs. PCOS. The data are presented as the mean ±SD (n = 3).

In addition, activation of AKT by SFP effectively abrogated the TECOS-DMBG co-treatment-mediated inhibition of DNMT1 phosphorylation (Figure 5(b)) and nuclear translocation (Figure 5(c)). Moreover, SFP significantly decreased the increased H19 expression following co-treatment with TECOS and DMBG (Figure 5(a)). Together, these results indicated that TECOS and DMBG co-treatment induced H19 expression via suppressing the PI3K/AKT-DNMT1 pathway.

Figure 5.

Figure 5.

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Effect of AKT activation by SFP on levels of H19 and PI3K/AKT-DNMT1 pathway-related proteins following TECOS-DMBG co-treatment.

Rat ovarian granulosa cells were divided into PCOS (stimulated with 1.0 μmol/L insulin for 48 h), and TECOS (sitagliptin) + DMBG (metformin) + SC79 (an AKT specific activator) group. (a) H19 expression was examined using qRT-PCR. (b) The protein levels of p-DNMT1 and DNMT1 were examined using western blot. (c) The nuclear translocation of DNMT1 was analyzed under a fluorescence microscope. The green signals indicated DNMT1 and the cell nuclei were labeled in blue by DAPI. Scale bar: 50 μm. **p < 0.01, vs. PCOS; ##p < 0.01, vs. TECOS + DMBG. The data are presented as the mean ±SD (n = 3).

H19 upregulation was involved in the TECOS-DMBG co-treatment-mediated improvement of reproductive hormone disorders, ovarian polycystic changes, and IR in PCOS rats

Finally, we assessed the in vivo effect of TECOS-DMBG co-treatment on reproductive hormone disorders, ovarian polycystic changes, and IR in experimental PCOS rats. Compared with the normal rats, PCOS rats showed significantly increased serum levels of LH, E2, T, and P but decreased levels of FSH (Table 1). Furthermore, PCOS rats showed increased serum levels of FBG, FINS, and HOMA-IR (Table 2), but decreased protein levels of IRS-1 and GLUT4 in ovarian tissues (Figure 6(c)). Moreover, HE staining revealed that the number of granulosa cells was significantly diminished in the model group. The normal rats displayed more complete corpora lutea and different levels of follicles with a thickening layer of granulosa cells (Figure 6(a)). Co-treatment with TECOS and DMBG improved reproductive hormone disorders (Table 1), ovarian polycystic changes (Figure 6(a)), and IR (Table 2 and Figure 6(c)) of PCOS rats.

Table 1.

Levels of LH, E2, FSH, T, and P in rats in each group.

Groups LH (IU/L) E2 (mU/L) FSH (IU/L) T (nmol/) P (nmol/L)
Normal
PCOS
TECOS+DMBG
TECOS+DMBG+LV-NC
TECOS+DMBG+LV-H19		 2.11 ± 0.07
6.73 ± 0.84**		 74.10 ± 8.15
112.36 ± 9.3**		 2.17 ± 0.07
1.10 ± 0.02**		 0.31 ± 0.02
0.52 ± 0.04**		 8.91 ± 0.85
26.24 ± 2.76**
3.50 ± 0.29##
3.64 ± 0.43
2.31 ± 0.11& 		86.54 ± 6.32##
87.10 ± 5.76
76.41 ± 8.55		 2.03 ± 0.08##
2.18 ± 0.10
2.40 ± 0.07& 		0.35 ± 0.03##
0.37 ± 0.02
0.27 ± 0.03& 		17.91 ± 1.85##
16.53 ± 1.76
9.10 ± 0.86&&
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**p < 0.01 vs. Normal; ##p < 0.01 vs. PCOS; &p < 0.05, &&p < 0.01 vs. TECOS+DMBG+LV-NC. LH, luteotropic hormone; E2, estradiol; FSH, follicle-stimulating hormone; T, testosterone; P, progesterone.

Table 2.

Levels of FBG, FINS, and HOMA-IR in rats in each group.

Groups FBG (mmol/L) FINS (mU/L) HOMA-IR
Normal
PCOS
TECOS+DMBG
TECOS+DMBG+LV-NC
TECOS+DMBG+LV-H19		 3.54 ± 0.15 13.82 ± 1.42 2.04 ± 0.09
6.63 ± 0.47**
4.62 ± 0.56##
4.65 ± 0.41
3.39 ± 0.22& 		21.43 ± 2.30**
16.64 ± 0.87#
16.81 ± 1.11
12.04 ± 0.53& 		5.74 ± 0.40**
3.12 ± 0.13##
3.14 ± 0.11
1.70 ± 0.08&&
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**p < 0.01 vs. Normal; #p < 0.05, ##p < 0.01 vs. PCOS; &p < 0.05, &&p < 0.01 vs. TECOS+DMBG+LV-NC. FBS, fasting blood glucose; FINS, fasting insulin; HOMA-IR, homeostasis model assessment for insulin resistance.

Figure 6.

Figure 6.

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H19 upregulation was involved in the TECOS-DMBG co-treatment-mediated improvement of ovarian polycystic changes and IR in PCOS rats.

Female SD rats were randomly divided into five groups: Normal, PCOS, TECOS+DMBG, TECOS+DMBG+LV-NC, and TECOS+DMBG+LV-H19 group. (a) The morphological changes of ovarian tissues were evaluated by hematoxylin and eosin (HE) staining. Scale bar: 25 μm. (b) H19 expression in ovarian tissues was examined using qRT-PCR. (c) The protein levels of IRS-1, GLUT4, PI3K, AKT, p-AKT, DNMT1, and p-DNMT1 in ovarian tissues were examined using western blot. *p < 0.05, **p < 0.01 vs. Normal; #p < 0.05, ##p < 0.01 vs. PCOS; &p < 0.05, &&p < 0.01 vs. TECOS+DMBG+LV-NC. n = 8/group.

Furthermore, H19 expression was significantly decreased in the PCOS group than that in the normal group but increased by co-treatment with TECOS and DMBG (Figure 6(b)). In contrast, the protein levels of PI3K as well as phosphorylation levels of AKT and DNMT1 were significantly up-regulated in the PCOS group compared with the normal group but downregulated by co-treatment with TECOS and DMBG (Figure 6(c)). Moreover, upregulation of H19 by H19-expressing lentiviruses significantly enhanced the TECOS-DMBG co-treatment-mediated improvement of reproductive hormone disorders (Table 1), ovarian polycystic changes (Figure 6(a)) and IR (Table 2 and Figure 6(c)), as well as increase in protein levels of PI3K plus phosphorylation levels of AKT and DNMT1 (Figure 6(c)). These data indicated that H19 upregulation was involved in the mechanism by which TECOS-DMBG co-treatment improved reproductive hormone disorders, ovarian polycystic changes, and IR.

Discussion

The present study provided evidence that co-treatment with TECOS and DMBG attenuated the induced apoptosis and IR in insulin-induced PCOS model cells, and improved reproductive hormone disorders, ovarian polycystic changes, and IR in PCOS model rats. Mechanistically, upregulation of H19 enhanced the efficacy of combination therapy. Furthermore, co-treatment with TECOS and DMBG induced H19 expression via inhibiting PI3K/AKT-DNMT1 pathway.

DMBG is frequently used for patients with IR [15]. Furthermore, DMBG has an established role in the management of PCOS for improving the growth and development of follicles and endometrium, increasing the ovulation rates and restoring the menstrual cycle of PCOS patients [16–18]. Combined therapy demonstrates greater efficacy in treating PCOS-related anovulation than its individual components [5]. DMBG and pioglitazone combination therapy demonstrated great efficacy in ameliorating PCOS in estradiol valerate-induced PCOS rats [19]. However, pioglitazone is associated with an increased risk of bladder cancer when compared with other antidiabetic drugs [20]. Other side effects such as edema [21] and bone fractures [22] were also related to the use of pioglitazone. In a previous study, TECOS in adjunct to DMBG prevented weight regain more effectively than DMBG alone in obese women with PCOS previously treated with liraglutide [6]. Another study also showed that TECOS improved beta-cell function and prevented a conversion to impaired glucose tolerance and type 2 diabetes in DMBG-intolerant and obese PCOS patients [23]. A more recent study demonstrated that TECOS lowered fasting blood glucose, relieved the high androgen state of PCOS rats and delayed the process of ovarian fibrosis [24]. Currently, the efficacy for the combination therapy with DMBG and TECOS in PCOS remains unclear. In this study, we found that TECOS-DMBG co-treatment demonstrated greater efficacy in attenuating the induced granulosa cell apoptosis and IR in PCOS model cells than DMBG mono-treatment. Furthermore, DMBG-TECOS co-treatment improved reproductive hormone disorders, ovarian polycystic changes, and IR in PCOS rats.

Accumulating evidence has indicated the diverse mechanistic roles of lncRNA H19 in various cancers [25], endometriosis [26] and infertility [27]. In addition, H19 also plays an important role in postnatal β-cell mass expansion in rats and contributes to the mechanisms compensating for IR in obesity [28]. A more recent study showed that in vivo silencing of H19 in normal mice caused hyperglycemia, hyperinsulinemia and impaired glucose, insulin, and pyruvate tolerance [29]. IR is prevalent in PCOS women and is critically involved in the pathogenesis of PCOS [3,4]. These data therefore indicated the potential role of H19 in PCOS. The current study provided the first evidence that H19 expression was significantly decreased in PCOS model cells and PCOS rats, but increased by co-treatment with TECOS and DMBG. This was consistent with the results of a study showing that H19 expression was decreased in ovarian granulosa cells of PCOS patients [8]. Furthermore, overexpression of H19 enhanced this combination therapy effect on inhibiting granulosa cell apoptosis, and improving reproductive hormone disorders, ovarian polycystic changes, and IR in PCOS rats. Our findings suggested that co-treatment with TECOS and DMBG may ameliorate PCOS with IR, at least partially, through upregulation of H19.

PI3K/AKT signaling plays an important role in the pathogenesis of PCOS, such as IR, fat cell differentiation syndrome, and growth of ovarian follicles [30]. It was showed that the PI3K/AKT signaling pathway was activated in PCOS-IR rats, while metformin inhibited PI3K/AKT signaling pathway [12]. These results were consistent with the finding in this present study that the protein levels of PI3K and phosphorylation levels of AKT were significantly up-regulated but then decreased by TECOS-DMBG co-treatment in insulin-induced PCOS cells and PCOS rats. Activation of PI3K/AKT signaling pathway leads to phosphorylation of DNMT1, which increases DNMT1 nuclear translocation and activity, leading to methylation of downstream genes [11]. DNA methyltransferase (DNMT) proteins can catalyze the conversion of cytosine to 5-methylcytosine (5mC), altering the epigenetic state of DNA. Overexpression of DNMT1 mimics 5mC enrichment of H19 promoter and subsequently downregulates H19 expression [10]. Here, our results showed that activation of AKT by SFP treatment effectively abrogated the TECOS-DMBG co-treatment-mediated inhibition of DNMT1 phosphorylation and nuclear translocation plus induction of H19 expression. Therefore, these data together with our findings indicated that DMBG-TECOS co-treatment inhibited PI3K/AKT signaling pathway and subsequently suppressed DNMT1 phosphorylation and nuclear translocation, leading to suppression of H19 methylation and upregulation of H19 expression.

Conclusion

In conclusion, DMBG and TECOS combination therapy ameliorates PCOS with IR, at least partially, through upregulation of lncRNA H19. Furthermore, co-treatment with TECOS and DMBG induces H19 expression via inhibiting the PI3K/AKT-DNMT1 pathway.

Funding Statement

This study was financially supported by the key project of Science and Technology Department in Henan Province (No. 142102310413).

Disclosure statement

No potential conflict of interest was reported by the authors.

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