Hypothalamic Epac2/AKT/GnRH signalling is associated with metformin-responsive neuroendocrine improvement in a PCOS model

Polycystic ovary syndrome (PCOS) is a complex endocrine-metabolic disorder characterised by hypothalamic-pituitary-gonadal (HPG) axis dysfunction and pathological gonadotropin-releasing hormone (GnRH) hypersecretion. While metformin demonstrates therapeutic efficacy in PCOS, its potential neuroendocrine mechanisms remain incompletely understood. This study aimed to investigate the involvement of hypothalamic signalling in metformin-responsive neuroendocrine regulation in PCOS. Letrozole-induced PCOS rat models were treated with metformin to assess reproductive and metabolic outcomes. Data-independent acquisition (DIA) proteomics of hypothalamic tissue was performed to identify dysregulated pathways, followed by network analysis to explore candidate regulatory nodes. Mechanistic investigations were conducted in glucosamine-induced insulin-resistant GT1-7 GnRH neurons using pharmacological modulation and siRNA knockdown. In vivo functional relevance was examined using AAV-mediated Epac2 knockdown in the mouse arcuate nucleus. Metformin improved metabolic and reproductive abnormalities in PCOS rats, including insulin resistance, GnRH/LH hypersecretion, estrous cyclicity disruption, and ovarian morphological alterations. Hypothalamic proteomic and network analyses suggested that metformin-responsive changes were associated with insulin and cAMP-related signalling pathways. In GT1-7 neurons, metformin increased Epac2 expression, restored AKT signalling, and reduced GnRH overproduction under insulin-resistant conditions. Hypothalamic Epac2 knockdown reduced AKT signalling and was associated with systemic insulin resistance, increased GnRH/LH secretion, and preliminary supportive ovarian morphological observations, consistent with a functional contribution of Epac2 to reproductive-metabolic regulation. These findings support the involvement of hypothalamic Epac2/AKT/GnRH signalling in metformin-responsive neuroendocrine regulation in PCOS. Further studies are required to determine the relative contributions of central and peripheral mechanisms.