The molecular mechanism of insulin action in human theca and adipocyte cells in polycycstic ovarian syndrome

PCOS is one of the leading causes of infertility worldwide affecting 1 in 10 women of a reproductive age. One of the fundamental abnormalities in women with PCOS can be seen within hormonal irregularities, which may include hyperandrogenemia hyperinsulinemia and hyper secretion of luteinising hormone (LH); and it is hypothesised that a defect in steroid secreting ovarian theca cells is involved due to their contribution in non-PCOS hormonal synthesis. Hyperinsulinemia has been associated with hyper-androgenemia through in vitro studies of cultured PCOS theca, where it has been suggested that insulin increases progesterone and androstenedione secretion when compared to normal theca cells. Furthermore the augmented effects of LH and insulin have been seen to increase ovarian androgen synthesis in non-PCOS theca cultures whilst also increasing the expression of steroidogenic enzymes specific to the PI3-K pathway. Many theories exist toward the etiology of hyper androgenemia within PCOS. Very few approaches however, consider dysfunction in multiple tissue types that may contribute to hormonal imbalances. It is well established that an association between obesity and PCOS exists and it is often the first therapeutic target for re-establishing reproductive function in obese PCOS patients. Furthermore PCOS patients tend to show distinct gynoid body fat distribution, which is reported to aggravate PCOS symptoms. It was therefore valid to examine the involvement in adipocyte function and its contribution to androgen levels within peos. This is further supported through the link between metabolic disorders such as insulin resistance and hyperinsulinemia, and their associations to obesity. Our study employed isolated preadipocyte and thecal cultures with close regulation of the influential factors LH and insulin. In doing so, we analysed androgen synthesis through activation and expression of steroidogenic enzymes CYP17 within both normal and polycystic ovaries. This allowed us to examine whether protein/hormonal concentrations vary across non-PCOS and peos cultures. This also allowed us to examine the possibility of a novel pathway leading to localised adipocyte synthesis as well as pinpointing whether dysfunction existed within the insulin-signalling pathway of thecal androgen steroidogenesis. The work in this thesis shows that adipocytes derived from non-PCOS and PCOS women, maintained in vitro differ on the basis of their morphology, rates of differentiation and proliferation. Furthermore, they reacted differently under conditions designed to mimic PCOS in vitro (increased insulin and LH), with reduced non-PCOS proliferation, and increased non-PCOS androgen secretion on insulin treatment. We also found increased steroidogenic CYP 17 expression in PCOS cultures under insulin stimulation. However PCOS adipocytes androstenedione secretion remained unaffected by insulin stimulation and secreted constant levels of androstenedione similar to that seen by insulin stimulated non-PCOS adipocytes. Our examination of non-PCOS and PCOS primary thecal cultures showed CYP17 expression is increased in pcas theca under basal conditions and that increases in insulin and LH leads to increases in in vitro theca proliferation. These conditions were also seen to lead to significant increases in androstenedione secretion over non-PCOS thecal cultures, and the results suggest it to be acting through the PI3-K pathway. These results therefore point to a specific area of dysfunction that should be further targeted for examination. Furthermore, they suggest that an adipocyte dysfunction exists within PCOS patients that may significantly contribute to hyperandrogenemia through localized synthesis of androgens

The molecular mechanism of insulin action in human theca and adipocyte cells in polycycstic ovarian syndrome

PCOS is one of the leading causes of infertility worldwide affecting 1 in 10 women of a reproductive age. One of the fundamental abnormalities in women with PCOS can be seen within hormonal irregularities, which may include hyperandrogenemia hyperinsulinemia and hyper secretion of luteinising hormone (LH); and it is hypothesised that a defect in steroid secreting ovarian theca cells is involved due to their contribution in non-PCOS hormonal synthesis. Hyperinsulinemia has been associated with hyper-androgenemia through in vitro studies of cultured PCOS theca, where it has been suggested that insulin increases progesterone and androstenedione secretion when compared to normal theca cells. Furthermore the augmented effects of LH and insulin have been seen to increase ovarian androgen synthesis in non-PCOS theca cultures whilst also increasing the expression of steroidogenic enzymes specific to the PI3-K pathway. Many theories exist toward the etiology of hyper androgenemia within PCOS. Very few approaches however, consider dysfunction in multiple tissue types that may contribute to hormonal imbalances. It is well established that an association between obesity and PCOS exists and it is often the first therapeutic target for re-establishing reproductive function in obese PCOS patients. Furthermore PCOS patients tend to show distinct gynoid body fat distribution, which is reported to aggravate PCOS symptoms. It was therefore valid to examine the involvement in adipocyte function and its contribution to androgen levels within peos. This is further supported through the link between metabolic disorders such as insulin resistance and hyperinsulinemia, and their associations to obesity. Our study employed isolated preadipocyte and thecal cultures with close regulation of the influential factors LH and insulin. In doing so, we analysed androgen synthesis through activation and expression of steroidogenic enzymes CYP17 within both normal and polycystic ovaries. This allowed us to examine whether protein/hormonal concentrations vary across non-PCOS and peos cultures. This also allowed us to examine the possibility of a novel pathway leading to localised adipocyte synthesis as well as pinpointing whether dysfunction existed within the insulin-signalling pathway of thecal androgen steroidogenesis. The work in this thesis shows that adipocytes derived from non-PCOS and PCOS women, maintained in vitro differ on the basis of their morphology, rates of differentiation and proliferation. Furthermore, they reacted differently under conditions designed to mimic PCOS in vitro (increased insulin and LH), with reduced non-PCOS proliferation, and increased non-PCOS androgen secretion on insulin treatment. We also found increased steroidogenic CYP 17 expression in PCOS cultures under insulin stimulation. However PCOS adipocytes androstenedione secretion remained unaffected by insulin stimulation and secreted constant levels of androstenedione similar to that seen by insulin stimulated non-PCOS adipocytes. Our examination of non-PCOS and PCOS primary thecal cultures showed CYP17 expression is increased in pcas theca under basal conditions and that increases in insulin and LH leads to increases in in vitro theca proliferation. These conditions were also seen to lead to significant increases in androstenedione secretion over non-PCOS thecal cultures, and the results suggest it to be acting through the PI3-K pathway. These results therefore point to a specific area of dysfunction that should be further targeted for examination. Furthermore, they suggest that an adipocyte dysfunction exists within PCOS patients that may significantly contribute to hyperandrogenemia through localized synthesis of androgens

Mechanisms of Luteinising Hormone Regulation in Female Steroidogenesis

Abstract Understanding the mechanisms of steroidogenic regulation in female fertility is fundamental to determining dysfunction. Research has revealed a strong relationship between fertility and metabolic disturbances. With many endocrine signals involved in activation of steroidogenic cascades, it has become necessary to map the complex cross-talk between these regulatory systems and this review looks to establish this based on current understanding. Luteininsing hormone is a vital endocrine hormone that is already known to signal via these pathways, although its involvement in metabolic disturbance is less understood. Studies investigating activation of the LH receptor in key steroidogenic cells, ovarian theca and granulosa, have highlighted overlaps in important signaling cascades including PKC/MAPK/PKA and PI3K. Here, we review LH and its signaling cross-talk in key steroidogenic pathways

Selective inhibition of plasma membrane calcium ATPase 4 improves angiogenesis and vascular reperfusion

Aims Ischaemic cardiovascular disease is a major cause of morbidity and mortality worldwide. Despite promising results from pre-clinical animal models, VEGF-based strategies for therapeutic angiogenesis have yet to achieve successful reperfusion of ischaemic tissues in patients. Failure to restore efficient VEGF activity in the ischaemic organ remains a major problem in current pro-angiogenic therapeutic approaches. Plasma membrane calcium ATPase 4 (PMCA4) negatively regulates VEGF-activated angiogenesis via inhibition of the calcineurin/NFAT signalling pathway. PMCA4 activity is inhibited by the small molecule aurintricarboxylic acid (ATA). We hypothesize that inhibition of PMCA4 with ATA might enhance VEGF-induced angiogenesis. Methods and results We show that inhibition of PMCA4 with ATA in endothelial cells triggers a marked increase in VEGF-activated calcineurin/NFAT signalling that translates into a strong increase in endothelial cell motility and blood vessel formation. ATA enhances VEGF-induced calcineurin signalling by disrupting the interaction between PMCA4 and calcineurin at the endothelial-cell membrane. ATA concentrations at the nanomolar range, that efficiently inhibit PMCA4, had no deleterious effect on endothelial-cell viability or zebrafish embryonic development. However, high ATA concentrations at the micromolar level impaired endothelial cell viability and tubular morphogenesis, and were associated with toxicity in zebrafish embryos. In mice undergoing experimentally-induced hindlimb ischaemia, ATA treatment significantly increased the reperfusion of post-ischaemic limbs. Conclusions Our study provides evidence for the therapeutic potential of targeting PMCA4 to improve VEGF-based pro-angiogenic interventions. This goal will require the development of refined, highly selective versions of ATA, or the identification of novel PMCA4 inhibitors

The molecular mechanism of insulin action in human theca and adipocyte cells in polycycstic ovarian syndrome

PCOS is one of the leading causes of infertility worldwide affecting I in 10 women of a reproductive age. One of the fundamental abnormalities in women with PCOS can be seen within hormonal irregularities, which may include hyperandrogenemia hyperinsulinemia and hyper secretion of luteinising hormone (LH); and it is hypothesised that a defect in steroid secreting ovarian theca cells is involved due to their contribution in non-PCOS hormonal synthesis. Hyperinsulinemia has been associated with hyper-androgenemia through in vitro studies of cultured PCOS theca, where it has been suggested that insulin increases progesterone and androstenedione secretion when compared to normal theca cells. Furthennore the augmented effects of LH and insulin have been seen to increase ovarian androgen synthesis in non-PCOS theca cultures whilst also increasing the expression of steroidogenic enzymes specific to the PI3-K pathway. Many theories exist toward the etiology of hyper androgenemia within PCOS. Very few approaches however, consider dysfunction in multiple tissue types that may contribute to hormonal imbalances. It is well established that an association between obesity and PCOS exists and it is often the first therapeutic target for re-establishing reproductive function in obese PCOS patients. Furthermore PCOS patients tend to show distinct gynoid body fat distribution, which is reported to aggravate PCOS symptoms. It was therefore valid to examine the involvement in adipocyte function and its contribution to androgen levels within PCOS. This is further supported through the link between metabolic disorders such as insulin resistance and hyperinsulinemia, and their associations to obesity. Our study employed isolated preadipocyte and thecal cultures with close regulation of the influential factors LH and insulin. In doing so, we analysed androgen synthesis through activation and expression of steroidogenic enzymes CYPl7 within both nonnal and polycystic ovaries. This allowed us to examine whether protein/hormonal concentrations vary across non-PCOS and PCOS cultures. This also allowed us to examine the possibility of a novel pathway leading to localised adipocyte synthesis as well as pinpointing whether dysfunction existed within the insulin-signalling pathway of thecal androgen steroidogenesis. The work in this thesis shows that adipocytes derived from non- PCOS and PCOS women, maintained in vitro differ on the basis of their morphology, rates of differentiation and proliferation. Furthennore, they reacted differently under conditions designed to mimic PCOS in vitro (increased insulin and LH), with reduced non- PCOS proliferation, and increased non- PCOS androgen secretion on insulin treatment. We also found increased steroidogenic CYPl7 expression in PCOS cultures under insulin stimulation. However PCOS adipocytes androstenedione secretion remained unaffected by insulin stimulation and secreted constant levels of androstenedione similar to that seen by insulin stimulated non-PCOS adipocytes. Our examination of non-PCOS and PCOS primary thecal cultures showed CYPl7 expression is increased in PCOS theca under basal conditions and that increases in insulin and LH leads to increases in in vitro theca proliferation. These conditions were also seen to lead to significant increases in androstenedione secretion over non-PCOS thecal cultures, and the results suggest it to be acting through the PI3-K pathway. These results therefore point to a specific area of dysfunction that should be further targeted for examination. Furthermore, they suggest that an adipocyte dysfunction exists within PCOS patients that may significantly contribute to hyperandrogenemia through localized synthesis of androgens.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Female adipocyte androgen synthesis and the effects of insulin

The metabolic syndrome is a cluster of metabolic disorders characterized by insulin resistance and hyperinsulinaemia, and its presence can increase the risk of cardiovascular disease significantly. The metabolic syndrome is associated with increased circulating androgen levels in women, which may originate from the ovaries and adrenal glands. Adipocytes are also able to synthesise steroid hormones, and this output has been hypothesised to increase with elevated insulin plasma concentrations. However, the contribution of the adipocytes to the circulating androgen levels in women with metabolic syndrome is limited and the effects of insulin are not fully understood. The aim of this study was to investigate the presence of steroid precursors and synthetic enzymes in human adipocyte biopsies as markers of possible adipocyte androgen synthesis. We examined pre and mature adipocytes taken from tissue biopsies of abdominal subcutaneous adipose tissue of participating women from the Department of Obstetrics and Gynaecology, of the Royal Derby Hospital. The results showed the potential for localised adipocyte androgen synthesis through the presence of the androgen precursor progesterone, as well as the steroid-converting enzyme 17α-hydroxylase. Furthermore, we found the controlled secretion of androstenedione in vitro and that insulin treatment caused levels to increase. Continued examination of a localised source of androgen production is therefore of clinical relevance due to its influence on adipocyte metabolism, its negative impact on female steroidogenic homeostasis, and the possible aggravation this may have when associated to obesity and obesity related metabolic abnormalities such as hyperinsulinaemia

Effects of rosa mosqueta oil supplementation in lipogenic markers associated with prevention of liver steatosis

© The Royal Society of Chemistry. Rosa mosqueta (RM) oil is rich in α-linolenic acid (ALA)-a precursor of eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), and it has a high antioxidant activity due to its abundant content of tocopherols. Additionally, it has been observed that RM oil administration prevents hepatic steatosis. Thus, the aim of this study was to demonstrate the antilipogenic mechanism related to RM oil administration in a high-fat diet (HFD) fed mice model by evaluating markers associated with the regulation of lipid droplet metabolism (PLIN2, PLIN5 and PPAR-γ), and proteins associated with lipogenesis (FAS and SREBP-1c). C57BL/6J mice were fed either a control diet or a HFD, with and without RM oil supplementation for 12 weeks. The results showed that RM oil supplementation decreases hepatic PLIN2 and PPAR-γ mRNA expression and SREBP-1c, FAS and PLIN2 protein levels, whereas we did not find changes in the level of PLIN5 among the groups. These results suggest tha

Reproductive and metabolic determinants of granulosa cell dysfunction in normal-weight women with polycystic ovary syndrome

ObjectiveTo determine the degree to which E2 hyperresponsiveness to FSH and antimüllerian hormone (AMH) overproduction in normal-weight women with polycystic ovary syndrome (PCOS) correlate with increased antral follicle number (AFN), hyperandrogenism, and/or metabolic dysfunction.DesignProspective cohort study.SettingAcademic medical center.Patient(s)Seven normal-weight women with PCOS (1990 National Institutes of Health criteria) ages 20-34 years and 13 age- and body mass index- (BMI-; 18.5-25 kg/m2) matched normoandrogenic ovulatory women were studied.Intervention(s)All women underwent basal serum hormone and metabolic measurements, FSH stimulation testing with transvaginal ovarian sonography, frequently sampled IV glucose tolerance testing, and whole-body dual-energy x-ray absorptiometry.Main outcome measure(s)Serum hormone/metabolite levels, 24-hour serum E2 response to 150 IU recombinant human (rh) FSH infusion, AFN, insulin sensitivity, and body mass measurements.Result(s)Serum E2 responsiveness to rhFSH and AMH levels were greater in women with PCOS than in BMI- and age-matched control women, as were serum androgen levels, AFN, and abdominal fat mass. In all women combined, serum E2 responsiveness to rhFSH was associated with AFN. Serum AMH levels, however, positively correlated with AFN but remained positively correlated with serum LH and free T levels and negatively correlated with total body fat and percent body fat, adjusting for AFN.Conclusion(s)In normal-weight women with PCOS, serum E2 hyperresponsiveness to rhFSH represents increased AFN, while elevated serum AMH levels reflect opposing effects of stimulatory reproductive (hyperandrogenism and increased AFN) versus inhibitory metabolic (body fat) factors. Given the small number of subjects reported, additional follow-up studies are required to confirm these data