Abstract
Purpose :
Clinical studies have shown that acute intensive insulin therapy causes a transient worsening of diabetic retinopathy in patients with type 1 and type 2 diabetes. The driving factors that regulate early worsening are unclear. There is controversy in the literature as to whether early worsening is a direct effect of insulin or a consequence of rapid and large reductions in blood glucose levels. We have previously demonstrated a direct effect of insulin on RPE and endothelial barrier disruption. In this study we test the hypothesis that insulin mediates disruption of the outer blood retinal barrier by disrupting claudin-19 tight junctions in the RPE.
Methods :
To visualize outer blood retinal barrier breakdown (oBRB), we developed a transgenic zebrafish line tg(RPE65:Claudin19-eGFP) which expresses claudin-19 fused to an enhanced green fluorescent protein in the retinal pigment epithelium (RPE). Male and female zebrafish larvae were treated from 3 to 6 days post fertilization with 8 units of insulin, 80 mM glucose, a combination of 8 units of insulin and 80 mM glucose or zebrafish water. The larvae eyes were then dissected, and immunohistochemistry was performed to visualize breakdown of the outer BRB. In addition, in vitro approaches with primary porcine RPE cells were used to study the effects of glucose/insulin on claudin-19 expression with RT-qPCR, western blots, and immunohistochemistry. Barrier function was evaluated by longitudinal impedance spectroscopy.
Results :
Zebrafish larvae exposed to insulin show breakdown of claudin-19 tight junctions and disruption of the outer blood retinal barrier. Primary porcine RPE cells treated with insulin showed a decrease in claudin-19 as well as reduced trans-epithelial electrical resistance which is indicative of loss of barrier function.
Conclusions :
Insulin induces outer blood retinal barrier breakdown regardless of the glucose concentration. These results provide insights into the molecular mechanisms regulating early worsening in DR as well as type-2 diabetes associated with hyperinsulinemia.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.