High glucose with insulin signalling blockade promotes cholesterol-sensitive, FAK-dependent UPEC invasion and blunts...

Diabetes increases susceptibility to urinary tract infection, but the epithelial mechanisms linking high glucose and insulin signalling blockade to UPEC invasion and weakened bladder defence remain incompletely defined. Here, we tested whether high glucose combined with insulin signalling blockade creates a bladder epithelial state that favours UPEC entry. In 5637 bladder epithelial cells, OSI-906 reduced AKT phosphorylation and, under high-glucose conditions, increased UPEC adhesion and intracellular recovery. This phenotype was associated with an increase in CTB/GM1-positive cells, consistent with membrane reorganisation toward more accessible GM1-enriched, cholesterol-sensitive domains that may provide permissive platforms for UPEC entry. Cholesterol perturbation with methyl-β-cyclodextrin reduced both the CTB/GM1 phenotype and UPEC invasion, whereas FAK inhibition with Y15 reduced invasion, supporting a cholesterol-sensitive membrane entry process that requires FAK activity. The relevance of these observations was examined in acute OSI-906-treated mice and chronically diabetic db/db mice. Acute OSI-906 treatment increased bladder UPEC burden and reduced selected antimicrobial-defence mediators. In db/db mice, increased bladder bacterial burden was accompanied by reduced early infection-associated host-defence responses compared with db/+ controls. Together, these findings suggest that high glucose, combined with insulin signalling blockade, reorganises the urothelial membrane toward a more accessible, cholesterol-sensitive state that favours FAK-dependent UPEC invasion while weakening bladder antimicrobial defence.