This two-year individual NRSA training fellowship application outlines the training and research plans for Dr. Danalea Skarra. Dr. Skarra has designed an integrated and intensive training program in the area of reproductive neuroendocrinology and metabolic regulation of fertility. In particular, Dr. Skarra proposes to study the integration between FOXO1 inhibition of the luteinizing hormone b subunit (Lhb) promoter and insulin signaling regulation of FOXO1 in pituitary gonadotropes. The proposed work and unique training environment at the University of California, San Diego will allow Dr. Skarra to achieve her long-term goal of becoming an independent academic researcher in the field of reproductive endocrinology, studying how metabolic disorders impact fertility. Training and development will entail mentoring by Dr. Varykina Thackray (Sponsor), an expert in the field of reproductive endocrinology, and Dr. Jerrold Olefsky (Co-Sponsor), a world-renowned researcher of insulin and glucose metabolism. Furthermore, she has established a Mentoring Committee comprised of Drs. Thackray and Olefsky, as well as Dr. Alexander Kauffman, a reproductive neuroendocrine expert, Dr. Mark Lawson, an expert in GnRH signaling, and FOXO1 expert Dr. Karen Arden. Dr. Skarra's comprehensive training plan is based on an intensive research experience, didactic instruction, training in responsible conduct of research, and acquisition of critical career skills. The research component of this proposal tests the hypothesis that insulin inhibition of FOXO1 increases Lhb transcription and LH production. Dr. Thackray's laboratory has reported that FOXO1 suppresses basal and GnRH-induced Lhb synthesis, and FOXO1's suppression localized to -150/-1 of the Lhb promoter. This region has been found by other researchers to be responsible for insulin induction of Lhb synthesis, potentially through EGR1. Furthermore, we can detect insulin-induced phosphorylation and subcellular localization changes of FOXO1 in pituitary gonadotropes, which is consistent with insulin's inhibitory effect on FOXO1 in other tissues.
In Aim 1, we will determine if FOXO1 suppresses EGR1 activity, thereby suppressing Lhb transcription, using luciferase assays, quantitative RT-PCR and western blot.
In Aim 2, primary pituitary cells will be used to investigate the effects of increased FOXO1 activity or ablation of FOXO1 on Lhb mRNA by quantitative RT-PCR and LH protein quantitation by immunoradiometric assay.
Aim 3 investigates the in vivo regulation of insulin on FOXO1 phosphorylation and subcellular localization in pituitaries from lean and diet-induced obese mice. Results from this proposal have the potential to identify mechanisms of infertility related to PCOS and obesity at the level of the pituitary gonadotrope, and it may provide insight into how the use of potential FOXO1-modulating drug treatments for diabetes, benign tumors, and cancer could impact Lhb production.
Obesity is a health epidemic and is associated with reproductive disorders in women, including menstrual irregularities, polycystic ovarian syndrome (PCOS), and infertility. Hyperinsulinemia and insulin resistance, which are frequently present in obesity and PCOS, are associated with adverse changes in hormones necessary for fertility, such as luteinizing hormone. This research investigates how insulin affects the production of luteinizing hormone, via the FOXO1 transcription factor, and may provide insight into the etiology and treatment of PCOS and obesity-related infertility.
