Insulin relaxes bladder via PI3K/AKT/eNOS pathway activation in mucosa: unfolded protein response-dependent insulin resistance as a cause of obesity-associated overactive bladder

Key points • Metabolic syndrome, obesity and insulin resistance are risk factors for overactive bladder, but little is known about the impact of insulin resistance on bladder functioning. • We aimed to investigate the insulin effects in murine and human bladder, and whether its defective action contributes to the bladder dysfunction associated with obesity. • Under physiological conditions, insulin relaxes urinary bladder through the release of nitric oxide from the mucosal layer, and this mechanism is impaired in bladders from insulin-resistant obese mice. • The defective insulin action in bladder mucosa from obese mice is due to endoplasmic reticulum stress, which remarkably contributes to the bladder overactivity in obesity conditions. • Our results enable a better understanding of the mechanism of action of insulin in the urinary bladder and how its defective action in mucosa contributes to bladder dysfunction in conditions of obesity-associated insulin resistance. Abstract We aimed to investigate the role of insulin in the bladder and its relevance for the development of overactive bladder (OAB) in insulin-resistant obese mice. Bladders from male individuals who were involved in multiple organ donations were used. C57BL6/J mice were fed with a high-fat diet for 10 weeks to induce insulin-resistant obesity. Concentration–response curves to insulin were performed in human and mouse isolated mucosa-intact and mucosa-denuded bladders. Cystometric study was performed in terminally anaesthetized mice. Western blot was performed in bladders to detect phosphorylated endothelial NO synthase (eNOS) (Ser1177) and the phosphorylated protein kinase AKT (Ser473), as well as the unfolded protein response (UPR) markers TRIB3, CHOP and ATF4. Insulin (1–100 nm) produced concentration-dependent mouse and human bladder relaxations that were markedly reduced by mucosal removal or inhibition of the PI3K/AKT/eNOS pathway. In mouse bladders, insulin produced a 3.0-fold increase in cGMP levels (P < 0.05) that was prevented by PI3K/AKT/eNOS pathway inhibition. Phosphoinositide 3-kinase (PI3K) inhibition abolished insulin-induced phosphorylation of AKT and eNOS in bladder mucosa. Obese mice showed greater voiding frequency and non-voiding contractions, indicating overactive detrusor smooth muscle. Insulin failed to relax the bladder or to increase cGMP in the obese group. Insulin-stimulated AKT and eNOS phosphorylation in mucosa was also impaired in obese mice. The UPR markers TRIB3, CHOP and ATF4 were increased in the mucosa of obese mice. The UPR inhibitor 4-phenyl butyric acid normalized all the functional and molecular parameters in obese mice. Our data show that insulin relaxes human and mouse bladder via activation of the PI3K/AKT/eNOS pathway in the bladder mucosa. Endoplasmic reticulum stress-dependent insulin resistance in bladder contributes to OAB in obese mice.