Elucidating the Role of Insulin‐induced AKR1C3 and the 11‐oxygenated Androgens in Promoting the Androgen Excess of Polycystic Ovary Syndrome (PCOS)

Abstract

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age. The androgen excess (AE) of PCOS is believed to promote cardio-metabolic risk, but how this occurs is unknown. As a result, treatment of cardio-metabolic diseases in PCOS women is not entirely effective. AE correlates with insulin resistance (IR) in these women and may be driving lipotoxicity. Aldo-keto reductase family 1 member C3 (AKR1C3) is induced by insulin and may be central in driving the AE of PCOS. Our goal is to understand the role of insulin-induced AKR1C3 in promoting AE and lipotoxicity in PCOS adipocytes. AKR1C3 predominantly catalyzes the conversion of peripheral androgens such Δ4-androstene-3,17-dione (4AD) to testosterone (T) and 5α-androstan-3,17-dione (5AD) to 5α-dihydrotestosterone (DHT). T and DHT are potent agonists for the androgen receptor (AR), which upregulate de novo lipogenesis. Fatty acid synthase (FASN) is known to be induced by androgens and may be responsible for this altered lipid profile in mature adipocytes. AKR1C3 overexpression in adipocytes, in response to insulin, could increase AR signaling and increase de novo lipogenesis. This lipid overflow could exacerbate insulin resistance through a feedforward mechanism and promote the lipotoxic profile seen in PCOS. Additionally, the adrenal 11-oxygenated androgens may be an alternative source of AE and have been reported as the dominant androgens of PCOS. We hypothesize that insulin induction of AKR1C3 and the conversion of 11-oxygenated androgens into potent AR agonists in adipocytes may potentiate AE and lipotoxicity in PCOS. Using differentiated Simpson-Golabi-Behmel Syndrome (SGBS) adipocytes as a model, we have shown that insulin increased AKR1C3 expression by >600 fold at the RNA level. Using pharmacological inhibition, we have preliminary data that shows insulin induction of AKR1C3 to utilize the PI3K/AKT/mTOR pathway as measured by RT-qPCR and immunoblot assays. Using a reverse-phase-HPLC discontinuous assay and post-reaction derivatization with dinitrophenylhydrazine, we determined the kcat and Km for the conversion of 11-ketoandrostene-3,17-dione (11K-4AD) to 11-ketotestosterone (11K-T) and 11-ketoandrostan-3,17-dione (11K-5AD) to 11-ketodihydrotestosterone (11K-DHT) using recombinant AKR1C3. We find that 11K-4AD and 11K-5AD are superior substrates for AKR1C3 when compared to 4AD and 5AD. Furthermore, we have quantified the formation of 11K-T in SGBS cells by stable-isotope-dilution (SID)-LC-MS/MS. This research will elucidate the mechanism of how AKR1C3 potentiates androgen excess and AR signaling to promote lipotoxicity in PCOS. Additionally, this work supports AKR1C3 as a potential therapeutic target in mitigating the androgen excess of PCOS.