Women with polycystic ovary syndrome (PCOS) have reproductive and metabolic abnormalities that result in an increased risk of infertility, diabetes and cardiovascular disease. The large intestine contains a complex community of microorganisms (the gut microbiome) that is dysregulated in humans with obesity and type 2 diabetes. Using a letrozole-induced PCOS mouse model, we demonstrated significant diet-independent changes in the gut microbial community, suggesting that gut microbiome dysbiosis may also occur in PCOS women. Letrozole treatment was associated with a time-dependent shift in the gut microbiome and a substantial reduction in overall species and phylogenetic richness. Letrozole treatment also correlated with significant changes in the abundance of specific Bacteroidetes and Firmicutes previously implicated in other mouse models of metabolic disease in a time-dependent manner. Our results suggest that the hyperandrogenemia observed in PCOS may significantly alter the gut microbiome independently of diet.

Ser/Thr protein phosphatase 5 (PP5) regulates several signaling-cascades that suppress growth and/or facilitate apoptosis in response to genomic stress. The expression of PP5 is responsive to hypoxia inducible factor-1 (HIF-1) and estrogen, which have both been linked to the progression of human breast cancer. Still, it is not clear if PP5 plays a role in the development of human cancer. Here, immunostaining of breast cancer tissue-microarrays (TMAs) revealed a positive correlation between PP5 over-expression and ductal carcinoma in situ (DCIS; P value 0.0028), invasive ductal carcinoma (IDC; P value 0.012) and IDC with metastases at the time of diagnosis (P value 0.0001). In a mouse xenograft model, the constitutive over-expression of PP5 was associated with an increase in the rate of tumor growth. In a MCF-7 cell culture model over-expression correlated with both an increase in the rate of proliferation and protection from cell death induced by oxidative stress, UVC-irradiation, adriamycin, and vinblastine. PP5 over-expression had no apparent effect on the sensitivity of MCF-7 cells to taxol or rapamycin. Western analysis of extracts from cells over-expressing PP5 revealed a decrease in the phosphorylation of known substrates for PP5. Together, these studies indicate that elevated levels of PP5 protein occur in human breast cancer and suggest that PP5 over-expression may aid tumor progression.

Background: Insulin signaling regulates luteinizing hormone (LH) production in pituitary gonadotrope cells through unknown mechanisms. Results: FOXO1 phosphorylation and cellular localization are regulated by insulin signaling, and FOXO1 represses LH ␤-subunit (Lhb) transcription. Conclusion: Our study highlights a novel mechanism for regulation of Lhb gene expression. Significance: FOXO1 may act as a metabolic sensor in controlling LH levels and fertility.

Women with polycystic ovary syndrome (PCOS) diagnosed with hyperandrogenism and ovulatory dysfunction have an increased risk of developing metabolic disorders, including type 2 diabetes and cardiovascular disease. We previously developed a model that uses letrozole to elevate endogenous testosterone levels in female mice. This model has hallmarks of PCOS, including hyperandrogenism, anovulation, and polycystic ovaries, as well as increased abdominal adiposity and glucose intolerance. In the current study, we further characterized the metabolic dysfunction that occurs after letrozole treatment to determine whether this model represents a PCOS-like metabolic phenotype. We focused on whether letrozole treatment results in altered pancreatic or liver function as well as insulin resistance. We also investigated whether hyperinsulinemia occurs secondary to weight gain and insulin resistance in this model or if it can occur independently. Our study demonstrated that letrozole-treated mice developed hyperinsulinemia after 1 week of treatment and without evidence of insulin resistance. After 2 weeks of letrozole treatment, mice became significantly heavier than placebo mice, demonstrating that weight gain was not required to develop hyperinsulinemia. After 5 weeks of letrozole treatment, mice exhibited blunted glucose-stimulated insulin secretion, insulin resistance, and impaired insulin-induced phosphorylation of AKT in skeletal muscle. Moreover, letrozole-treated mice exhibited dyslipidemia after 5 weeks of treatment but no evidence of hepatic disease. Our study demonstrated that the letrozole-induced PCOS mouse model exhibits multiple features of the metabolic dysregulation observed in obese, hyperandrogenic women with PCOS. This model will be useful for mechanistic studies investigating how hyperandrogenemia affects metabolism in females.

Synthesis of the gonadotropin β-subunits is tightly controlled by a complex network of hormonal signaling pathways that may be modulated by metabolic cues. Recently, we reported that insulin regulates FOXO1 phosphorylation and cellular localization in pituitary gonadotropes and that FOXO1 overexpression inhibits Lhb transcription. In the current study, we investigated whether FOXO1 modulates Fshb synthesis. Here, we demonstrate that FOXO1 represses basal and GnRH-induced Fshb transcription in LβT2 cells. In addition, we show that PI3K inhibition, which increases FOXO1 nuclear localization, results in decreased Fshb mRNA levels in murine primary pituitary cells. FOXO1 also decreases transcription from the human FSHB promoter, suggesting that FOXO1 regulation of FSHB transcription may be conserved between rodents and humans. Although the FOXO1 DNA-binding domain is necessary for suppression of Fshb, we do not observe direct binding of FOXO1 to the Fshb promoter, suggesting that FOXO1 exerts its effect through protein-protein interactions with transcription factors required for Fshb synthesis. FOXO1 suppression of basal Fshb transcription may involve PITX1 because PITX1 interacts with FOXO1, FOXO1 repression maps to the proximal Fshb promoter containing a PITX1-binding site, PITX1 induction of Fshb or a PITX1 binding element in CV-1 cells is decreased by FOXO1, and FOXO1 suppresses Pitx1 mRNA and protein levels. GnRH induction of an Fshb promoter containing a deletion at -50/-41 or -30/-21 is not repressed by FOXO1, suggesting that these two regions may be involved in FOXO1 suppression of GnRH-induced Fshb synthesis. In summary, our data demonstrate that FOXO1 can negatively regulate Fshb transcription and suggest that FOXO1 may relay metabolic hormonal signals to modulate gonadotropin production.

BackgroundA majority of women with polycystic ovary syndrome (PCOS) have metabolic dysfunction that results in an increased risk of type 2 diabetes. We previously developed a pubertal mouse model using the aromatase inhibitor, letrozole, which recapitulates many of the reproductive and metabolic features of PCOS. To further our understanding of the effects of androgen excess, we compared the effects of letrozole treatment initiated in puberty versus adulthood on reproductive and metabolic phenotypes as well as on the gut microbiome.ResultsLetrozole treatment of both pubertal and adult female mice resulted in reproductive hallmarks of PCOS, including hyperandrogenemia, anovulation and polycystic ovaries. However, unlike pubertal mice, treatment of adult female mice resulted in modest weight gain and abdominal adiposity, minimal elevation in fasting blood glucose and insulin levels, and no detectable insulin resistance. In addition, letrozole treatment of adult mice was associated with a distinct shift in gut microbial diversity compared to letrozole treatment of pubertal mice.ConclusionsOur results indicate that dysregulation of metabolism and the gut microbiome in PCOS may be influenced by the timing of androgen exposure. In addition, the minimal weight gain and lack of insulin resistance in adult female mice after letrozole treatment indicates that this model may be useful for investigating the effects of hyperandrogenemia on the hypothalamic-pituitary-gonadal axis and the periphery without the influence of substantial metabolic dysregulation.Electronic supplementary materialThe online version of this article (10.1186/s12866-019-1425-7) contains supplementary material, which is available to authorized users.

In the present study, we investigate whether the FOXO1 transcription factor modulates activin signaling in pituitary gonadotropes. Our studies show that overexpression of constitutively active FOXO1 decreases activin induction of murine Fshb gene expression in immortalized LβT2 cells. We demonstrate that FOXO1 suppression of activin induction maps to the −304/−95 region of the Fshb promoter containing multiple activin response elements and that the suppression requires the FOXO1 DNA-binding domain (DBD). FOXO1 binds weakly to the −125/−91 region of the Fshb promoter in a gel-shift assay. Since this region of the promoter contains a composite SMAD/FOXL2 binding element necessary for activin induction of Fshb transcription, it is possible that FOXO1 DNA binding interferes with SMAD and/or FOXL2 function. In addition, our studies demonstrate that FOXO1 directly interacts with SMAD3/4 but not SMAD2 in a FOXO1 DBD-dependent manner. Moreover, we show that SMAD3/4 induction of Fshb-luc and activin induction of a multimerized SMAD-binding element-luc are suppressed by FOXO1 in a DBD-dependent manner. These results suggest that FOXO1 binding to the proximal Fshb promoter as well as FOXO1 interaction with SMAD3/4 proteins may result in decreased activin induction of Fshb in gonadotropes.

The FOXO1 transcription factor is important for multiple aspects of reproductive function. We previously reported that FOXO1 functions as a repressor of gonadotropin hormone synthesis, but how FOXO1 is regulated in pituitary gonadotropes is unknown. The growth factors, insulin and insulin-like growth factor I (IGF1) function as key regulators of cell proliferation, metabolism and apoptosis in multiple cell types through the PI3K/AKT signaling pathway. In this study, we found that insulin and IGF1 signaling in gonadotropes induced FOXO1 phosphorylation through the PI3K/AKT pathway in immortalized and primary cells, resulting in FOXO1 relocation from the nucleus to the cytoplasm. Furthermore, insulin administration in vivo induced phosphorylation of FOXO1 and AKT in the pituitary. Thus, insulin and IGF1 act as negative regulators of FOXO1 activity and may serve to fine-tune gonadotropin expression.