MicroRNA‐21 Genetic Ablation Exacerbates Insulin Signaling Dysregulation in Hyperandrogenemic Female Mice

Abstract

IntroductionPolycystic Ovary Syndrome (PCOS) is the most common endocrine disorder in reproductive‐age women and is characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology. PCOS is associated with obesity and insulin resistance, both being risk factors associated with cardiovascular disease. Micro‐RNAs are small, non‐coding RNAs capable of altering gene expression by binding to specific mRNA targets. MicroRNA‐21(miR21) is cell‐specifically expressed and dynamically regulated in tissues that are implicated in cardiometabolic dysfunction in PCOS including the liver, skeletal muscle, and the heart. We hypothesize that miR‐21 genetic ablation exacerbates androgen‐induced dysregulation of insulin signaling to modify insulin resistance and cardiac damage in a mouse model of PCOS.MethodsThree‐week‐old peripubertal miR‐21 knockout (miR21KO) or wild‐type (WT) C57BL/6N female mice were treated s.c. with dihydrotestosterone (DHT, 8 mg) or vehicle for 90 days. Fasting glucose was determined by an enzymatic method and both fasting insulin and advanced glycation end products (AGES) were measured by ELISA. microRNA, mRNA, and protein levels were quantified by RT‐qPCR and Western‐blot. Data were analyzed by t‐test or two‐way ANOVA. All differences are considered significant p<0.05.ResultsIn WT animals DHT treatment increased miR21 expression in the soleus muscle (2.15±0.47‐fold, p<0.05), with a trend to increase in the tibialis anterior muscle (1.25‐fold), and the liver (1.41‐fold) compared with their vehicle counterparts. DHT‐treated miR21KO females showed exacerbated insulin resistance, having increased HOMA‐IR compared with both vehicle miR21KO and WT groups (6.50 ± 3.64 vs. 3.32±1.83 and 3.16±1.31, p<0.05) calculated using fasting insulin and glucose. In the soleus, miR21KO DHT‐treated mice showed an increase in Irs2 (1.45‐fold, p<0.05) compared with vehicle WT, whereas no increase was found in DHT‐treated WT animals. DHT‐treated miR21KO animals showed significantly increased Akt2 (1.40‐fold) and the glucose transporter Slc2a2 (1.24‐fold) mRNA expression in the liver. Additionally, liver protein expression of total mTOR was increased only in miR21KO DHT‐treated animals (2.40, p<0.05) compared to vehicle WT animals. Lastly, miR21KO increased left ventricular advanced glycation end products (AGEs), having significantly higher AGEs compared to all three treatment groups (3.10±0.52μg /mg protein, p<0.05).ConclusionsOur results suggest that miR21 upregulation in insulin sensitive tissues is a compensatory mechanism to counteract androgen‐inducted dysregulation of insulin signaling pathways and downstream cardiac damage. The differences observed in androgen‐treated mice highlight the potential importance of miR21 in the crosstalk between several insulin signaling networks. Notably, given the worsened DHT‐induced insulin resistance and increased left ventricular AGEs in miR21KO animals, a miR‐21 mimic may be a potential novel therapeutic option for women with PCOS.