Combined use of Diane-35 and metformin improves the ovulation in the PCOS rat model possibly via regulating glycolysis pathway.
ABSTRACT: BACKGROUND:Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disease with unknown pathogenesis. However, the treatment of Diane-35 combined with metformin can improve the endocrine and ovulation of PCOS. In this study, we investigated the effects of Diane-35 combined with metformin (DM) treatment on ovulation and glucose metabolism in a PCOS rat model. METHODS:Sprague Dawley rats were divided into 3 groups, control group, model group (PCOS group) and Diane-35 combined with metformin (PCOS + DM group). The mRNA expression levels were determined by qRT-PCR. The hormone levels were determined by enzyme-linked immunosorbent assay. Immunostaining detected the protein levels of lactate dehydrogenase A (LDH-A), pyruvate kinase isozyme M2 (PKM2) and sirtuin 1 (SIRT1) in the ovarian tissues. TNUEL assay was performed to determine cell apoptosis in the PCOS rats. The metabolites in the ovarian tissues were analyzed by liquid chromatography with tandem mass spectrometry. RESULTS:PCOS rats showed an increased in body weight, levels of luteinizing hormone and testosterone and insulin resistance, which was significantly attenuated by the DM treatment. The DM treatment improved disrupted estrous cycle and increased the granulosa cells of the ovary in the PCOS rats. The decreased proliferation and increased cell apoptosis of granulosa cells in the ovarian tissues of PCOS rats were significantly reversed by the DM treatment. The analysis of metabolics revealed that ATP and lactate levels were significantly decreased in PCOS rats, which was recovered by the DM treatment. Furthermore, the expression of LDH-A, PKM2 and SIRT1 was significantly down-regulated in ovarian tissues of the PCOS rats; while the DM treatment significantly increased the expression of LDH-A, PKM2 and SIRT1 in the ovarian tissues of the PCOS rats. CONCLUSION:In conclusion, our study demonstrated that Diane-35 plus metformin treatment improved the pathological changes in the PCOS rats. Further studies suggest that Diane-35 plus metformin can improve the energy metabolism of the ovary via regulating the glycolysis pathway. The mechanistic studies indicated that the therapeutic effects of Diane-35 plus metformin treatment in the PCOS rats may be associated with the regulation of glycolysis-related mediators including PKM2, LDH-A and SIRT1.
Project description:Polycystic ovary syndrome (PCOS) is a frequent endocrine and metabolic syndrome in reproductive-age women. Recently, emerging evidence has shown that gut microbiota is closely related to metabolic diseases such as type 2 diabetes, obesity and PCOS. In the present study, we established dihydrotestosterone (DHT)-induced PCOS rats and used Illumina MiSeq sequencing (PE300) to examine the composition, diversity, and abundance of the gut microbiota in PCOS. We compared the effects of three PCOS treatments: Diane-35 (estrogen and progesterone), probiotics and berberine. The DHT-induced rats showed constant estrous cycles, the loss of mature ovarian follicles, insulin resistance and obesity. The reproductive and metabolic functions in the PCOS rats were improved by treatment with Diane-35 and probiotics. Diane-35 and probiotics could restore the diversity of the gut microbiota, and the recovery of gut microbiota disorders improved the reproductive function in PCOS-like rats. However, berberine drastically reduced the species diversity and amount of gut microbiota and showed no improvement in PCOS. The findings of this study will help us to better understand the influence of the gut microbiota in the metabolic and reproductive alterations in PCOS as well as suggest opportunities for future personal dietary guidance for PCOS.
Project description:The underlying mechanisms of polycystic ovarian syndrome (PCOS)-induced endometrial dysfunction are not fully understood, and although accumulating evidence shows that the use of metformin has beneficial effects in PCOS patients, the precise regulatory mechanisms of metformin on endometrial function under PCOS conditions have only been partially explored. To address these clinical challenges, this study aimed to assess the protein expression patterns of glycolytic enzymes, estrogen receptor (ER), and androgen receptor (AR) along with differences in mitochondria-dependent apoptosis in PCOS patients with and without endometrial hyperplasia in vivo and to investigate the effects of metformin in PCOS patients with endometrial hyperplasia in vitro. Here, we showed that compared to non-PCOS patients and PCOS patients without hyperplasia, the endometria from PCOS patients with hyperplasia had a distinct protein expression pattern of glycolytic enzymes, including pyruvate kinase isozyme M2 isoform (PKM2) and pyruvate dehydrogenase (PDH), and mitochondrial transcription factor A (TFAM). In PCOS patients with endometrial hyperplasia, increased glandular epithelial cell secretion and infiltrated stromal cells in the glands were associated with decreased PDH immunoreactivity in the epithelial cells. Using endometrial tissues from PCOS patients with hyperplasia, we found that in response to metformin treatment in vitro, hexokinase 2 (HK2) expression was decreased, whereas phosphofructokinase (PFK), PKM2, and lactate dehydrogenase A (LDHA) expression was increased compared to controls. Although there was no change in PDH expression, metformin treatment increased the expression of TFAM and cleaved caspase-3. Moreover, our in vivo study showed that while endometrial ER? expression was no different between non-PCOS and PCOS patients regardless of whether or not hyperplasia was present, ER? and AR protein expression was gradually increased in women with PCOS following the onset of endometrial hyperplasia. Our in vitro study showed that treatment with metformin inhibited ER? expression without affecting ER? expression. Our findings suggest that decreased glycolysis and increased mitochondrial activity might contribute to the onset of ER?-dependent endometrial hyperplasia and that metformin might directly reverse impaired glycolysis and normalize mitochondrial function in PCOS patients with endometrial hyperplasia.
Project description:BACKGROUND:Rats with hyperandrogen-induced polycystic ovary syndrome (PCOS) have been shown to develop ovarian oxidative stress (OS) and fibrosis. The Sirt1 agonist, resveratrol, can reduce OS through inhibiting p66Shc in other models of OS. METHODS:We created a rat PCOS model with increased OS levels following treatment with one of the two androgens, dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT). The PCOS related features were determined by measurement of malondialdehyde (MDA) and superoxide dismutase (SOD) levels or by examining the reactive oxygen species (ROS) levels using the DCF-DA probe. The potential mechanisms by which p66Shc/Sirt1 mediates ovarian fibrosis were explored by western blotting, quantitative reverse transcription-PCR, immunofluorescence staining, and immunohistochemistry. RESULTS:Hyperandrogen dramatically augmented OS and activation of fibrotic factors in the ovary. Our data demonstrated that treatment with resveratrol enhanced Sirt1 and decreased ovarian OS as well as inhibited phosphorylation of p66Shc both in vivo and in vitro. The treatment suppressed fibrotic factor activation and improved ovarian morphology. Lentivirus- or siRNA-mediated p66Shc knockdown resulted in a dramatic enhancement of Sirt1 expression, down-regulation of ROS and suppression of fibrotic factors in granulosa cells. Moreover, p66Shc overexpression markedly increased the expression of fibrotic factors. Additionally, silencing Sirt1 induced a dramatic increase in p66Shc and enhanced activation of fibrotic factors. CONCLUSIONS:p66Shc may be a direct target of Sirt1 for inducing ROS and thus promoting fibrosis. Further exploration of the mechanisms of p66Shc in both fibrosis and OS may provide novel therapeutic strategies that will facilitate the improvement in PCOS symptoms and reproductive functions.
Project description:There is increasing proof that polycystic ovary syndrome (PCOS) is associated with the increased frequency of thyroid disturbances. Chamomile (Matricaria chamomilla L.) herb and metformin showed therapeutic efficacy against polycystic ovary syndrome (PCOS). This study aimed to investigate the possible therapeutic effect of both chamomile flower extract and metformin against thyroid damage associated with PCOS in rats. The PCOS model was developed in rats by injecting estradiol valerate, and it was confirmed to be associated with thyroid hypofunction biochemically and pathologically. Treatment of PCOS rats with both chamomile extract and metformin resulted in an improvement in serum level of thyroid hormones (TSH, p < 0.01; T3 and T4, p < 0.05) and the disappearance of most thyroid gland pathological changes demonstrated by light and electron microscopes. They also reduced the level of serum estrogen (p < 0.01). Both chamomile extract and metformin decreased MDA (p < 0.05) and increased GPx and CAT (p < 0.01). Only chamomile extract increased GSH (p < 0.01). Both treatments reduced the apoptotic death of thyroid cells as noted by the reduction of caspase-3 immunoexpression (p < 0.01). In conclusion, both Matricaria chamomilla extract and metformin ameliorated hypothyroidism associated with PCOS through an antioxidant and antiapoptotic mechanism.
Project description:BACKGROUND:Novel therapeutic approaches are required to treat ovarian cancer and dependency on glycolysis may provide new targets for treatment. This study sought to investigate the variation of expression of molecular components (GLUT1, HKII, PKM2, LDHA) of the glycolytic pathway in ovarian cancers and the effectiveness of targeting this pathway in ovarian cancer cell lines with inhibitors. METHODS:Expression of GLUT1, HKII, PKM2, LDHA were analysed by quantitative immunofluorescence in a tissue microarray (TMA) analysis of 380 ovarian cancers and associations with clinicopathological features were sought. The effect of glycolysis pathway inhibitors on the growth of a panel of ovarian cancer cell lines was assessed by use of the SRB proliferation assay. Combination studies were undertaken combining these inhibitors with cytotoxic agents. RESULTS:Mean expression levels of GLUT1 and HKII were higher in high grade serous ovarian cancer (HGSOC), the most frequently occurring subtype, than in non-HGSOC. GLUT1 expression was also significantly higher in advanced stage (III/IV) ovarian cancer than early stage (I/II) disease. Growth dependency of ovarian cancer cells on glucose was demonstrated in a panel of ovarian cancer cell lines. Inhibitors of the glycolytic pathway (STF31, IOM-1190, 3PO and oxamic acid) attenuated cell proliferation in platinum-sensitive and platinum-resistant HGSOC cell line models in a concentration dependent manner. In combination with either cisplatin or paclitaxel, 3PO (a novel PFKFB3 inhibitor) enhanced the cytotoxic effect in both platinum sensitive and platinum resistant ovarian cancer cells. Furthermore, synergy was identified between STF31 (a novel GLUT1 inhibitor) or oxamic acid (an LDH inhibitor) when combined with metformin, an inhibitor of oxidative phosphorylation, resulting in marked inhibition of ovarian cancer cell growth. CONCLUSIONS:The findings of this study provide further support for targeting the glycolytic pathway in ovarian cancer and several useful combinations were identified.
Project description:Advanced glycation end-products (AGEs) are involved in the pathogenesis and consequences of polycystic ovary syndrome (PCOS), a complex metabolic disorder associated with female infertility. The most powerful AGE precursor is methylglyoxal (MG), a byproduct of glycolysis, that is detoxified by the glyoxalase system. By using a PCOS mouse model induced by administration of dehydroepiandrosterone (DHEA), we investigated whether MG-dependent glycative stress contributes to ovarian PCOS phenotype and explored changes in the Sirtuin 1 (SIRT1) functional network regulating mitochondrial functions and cell survival. In addition to anovulation and reduced oocyte quality, DHEA ovaries revealed altered collagen deposition, increased vascularization, lipid droplets accumulation and altered steroidogenesis. Here we observed increased intraovarian MG-AGE levels in association with enhanced expression of receptor for AGEs (RAGEs) and deregulation of the glyoxalase system, hallmarks of glycative stress. Moreover, DHEA mice exhibited enhanced ovarian expression of SIRT1 along with increased protein levels of SIRT3 and superoxide dismutase 2 (SOD2), and decreased peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC1?), mitochondrial transcriptional factor A (mtTFA) and translocase of outer mitochondrial membrane 20 (TOMM20). Finally, the presence of autophagy protein markers and increased AMP-activated protein kinase (AMPK) suggested the involvement of SIRT1/AMPK axis in autophagy activation. Overall, present findings demonstrate that MG-dependent glycative stress is involved in ovarian dysfunctions associated to PCOS and support the hypothesis of a SIRT1-dependent adaptive response.
Project description:Preclinical, early phase clinical trials and epidemiological evidence support the potential role of insulin-sensitizers in cancer prevention and treatment. Insulin-sensitizers improve the metabolic and hormonal profile in PCOS patients and may also act as anticancer agents, especially in cancers associated with hyperinsulinemia and oestrogen dependent cancers. Several lines of evidence support the protection against cancer exerted by dietary inositol, in particular inositol hexaphosphate. Metformin, thiazolidinediones, and myoinositol postreceptor signaling may exhibit direct inhibitory effects on cancer cell growth. AMPK, the main molecular target of metformin, is emerging as a target for cancer prevention and treatment. PCOS may be correlated to an increased risk for developing ovarian and endometrial cancer (up to threefold). Several studies have demonstrated an increase in mortality rate from ovarian cancer among overweight/obese PCOS women compared with normal weight women. Long-term use of metformin has been associated with lower rates of ovarian cancer. Considering the evidence supporting a higher risk of gynaecological cancer in PCOS women, we discuss the potential use of insulin-sensitizers as a potential tool for chemoprevention, hypothesizing a possible rationale through which insulin-sensitizers may inhibit tumourigenesis.
Project description:Polycystic ovary syndrome (PCOS) is characterized by failure of ovulation and is associated with obesity and chronic inflammation. Recent evidence suggests that anomalous activation of ovarian macrophages and numerical and functional deficits in the Th17 (CD4+IL17A+) and the CD4+CD25+CD127low Tregs plays crucial role in PCOS. We have shown that the pre-pregnancy use of tacrolimus prevents adverse reproductive outcomes in a mouse model of PCOS. Here we used the HFD-NONcNZO mice to test a hypothesized beneficial use of tacrolimus relative to metformin in favorably influencing the ovarian and systemic immune milieux conducive to gestational success in subjects with PCOS. Compared to normative controls, our data revealed an aberrant peri-conceptional suppression of the CD4+CD25+CD127low Tregs together with an overexpression of the Th17 T cells and lack of coordinated activation of ovarian macrophages in untreated HFD-dNONcNZO mice. Significant variances in treatment outcomes favoured the use of tacrolimus over metformin in treated mice. Consistent with the human fertility studies, this investigation reveals a beneficial systemic use of tacrolimus (0.1?mg/kg) in promoting early pregnancy in individuals with PCOS and suggests the need for further research into the selective inhibition of IL17A as a plausibly alternative immunotherapeutic approach in the clinical management of infertile individuals with PCOS.
Project description:Low-frequency electro-acupuncture (EA) has been shown to restore ovulation in patients with polycystic ovary syndrome (PCOS), and previous animal experiments showed that EA improves ovarian blood flow and angiogenesis. We performed EA for 4 weeks in dihydrotestosterone (DHT)-induced PCOS-like rats and investigated the three-dimensional (3D) ovarian innervation to determine the role of innervation in folliculogenesis and vascularity. Ovarian tissues were made transparent following the CUBIC 3D tissue-clearing protocol and were immunostained using antibodies against platelet endothelial cell adhesion molecule-1 and tyrosine hydroxylase to visualize the ovarian vasculature and innervation, respectively. This was followed by 3D imaging using lightsheet microscopy and analysis using the Imaris software. In control rats, ovarian innervation increased with age, and the neuronal branching started from the ovarian hilum and reached the individual follicles at different follicle stages. At the individual follicle level, each follicle was mainly innervated by one neuronal fiber. Compared with control rats, ovaries from DHT-treated PCOS-like rats had more antral follicles and fewer preovulatory follicles and corpora lutea. Furthermore, PCOS ovaries showed decreased innervation of blood vessels near the hilum and the surrounding individual antral follicles. EA in PCOS-like rats led to increased numbers of preovulatory follicles and corpora lutea together with increased innervation of blood vessels near the hilum. To determine the role of ovarian innervation, we further performed unilateral sectioning of the superior ovarian nerve (SON) in PCOS + EA rats and found that the left sectioned ovary had fewer preovulatory follicles and corpora lutea compared with those in the right non-sectioned ovary. In conclusion, ovarian innervation likely played an important role in folliculogenesis, and EA might restore PCOS pathophysiology by regulating ovarian innervation, at least partially mediated through the SON.
Project description:Ovarian cancer is a common gynecological cancer that is found worldwide. Class III histone deacetylase (HDAC) inhibitors, a new class of anticancer agents, induce autophagy in various human cancer cells. The aim of the present study was to investigate the antitumor activity of MHY2245, a new synthetic SIRT inhibitor, on human ovarian cancer cells. We found that MHY2245 exhibited potent cytotoxicity to SKOV3 cells in a time- and concentration-dependent manner. The cytotoxicity of MHY2245 (IC50=0.32 µM) was higher than that of doxorubicin (DOX, IC50=1.38µM) against SKOV3 cells. MHY2245 significantly inhibited SIRT1 enzyme activity, reduced the expression of SIRT1, increased cell cycle arrest at G2/M phase, and induced apoptotic cell death in SKOV3 cells via expression of cytochrome c, cleaved-PARP, cleaved caspase-3, and Bax. This might be associated with blocking of the pyruvate kinase M2 (PKM2)/mTOR pathway. MHY2245 also inhibited tumor growth and reduced tumor size when SKOV3 cells were transplanted into nude mice. Our results indicate that MHY2245 exerts antitumor activity against ovarian cancer cells by blocking the PKM2/mTOR pathway. We suggest that MHY2245 is a promising anticancer agent that disrupts ovarian cancer cell metabolism.