Insulin Induces Outer Blood–Retinal Barrier Disruption via Downregulation of Claudin-19

Purpose: Diabetic retinopathy is a common complication of diabetes mellitus, a disease that is reaching epidemic proportions worldwide. Although diabetic macular edema has generally been attributed to breakdown of the inner blood-retinal barrier, accumulating evidence suggests that the outer blood-retinal barrier (oBRB) may also be involved. Clinical studies have shown that acute intensive insulin therapy causes a transient worsening of diabetic retinopathy in type 1 and type 2 diabetes. In this study, we tested the hypothesis that insulin directly disrupts the oBRB by targeting claudin-19 tight junctions in the retinal pigment epithelium (RPE). Methods: The effects of insulin on claudin-19 tight junctions in primary RPE cells were assessed by immunohistochemistry and western blot analysis using in vitro cell culture models and an in vivo transgenic zebrafish model. Changes in blood-retinal barrier integrity were quantified using electric cell-substrate impedance sensing (ECIS). Results: Claudin-19 was identified as the predominant claudin in primary porcine RPE cells and was essential for maintaining oBRB integrity. Barrier function did not differ between RPE cells cultured under physiological (5 mM) or high (25 mM; diabetic) glucose conditions. In contrast, insulin treatment disrupted the oBRB independently of glucose concentration. Insulin significantly reduced claudin-19 protein levels without affecting transcript abundance, indicating post-transcriptional regulation. Consistent with the in vitro findings, insulin induced claudin-19 tight junction disruption in vivo in a transgenic zebrafish model expressing claudin-19 fused to enhanced green fluorescent protein. Consistent with the in vitro findings, high glucose alone did not disrupt claudin-19 in vivo. Conclusions: These findings demonstrate that insulin disrupts the oBRB independently of glucose concentration in both in vitro and in vivo models. This work provides new insight into the molecular mechanisms underlying early worsening of diabetic retinopathy and highlights a potential role for hyperinsulinemia in type 2 diabetes-associated retinal pathology.