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Laura P. Pardon, Steven B. Koevary; Effects of Insulin Eye Drops on pAkt and eNOS Expression in the Rat. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3253.
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We previously reported that insulin applied topically in the rat accumulates in the retina, and preliminary data suggest that such treatment short-term might be beneficial in preventing background retinopathy in diabetic rats. Before undertaking a long-term study in diabetic rats to determine whether insulin eye drop treatment can indeed prevent the development of microaneurysms, acellular capillaries, and pericyte loss, it would be important to show that insulin accumulating in the retina following topical application can influence the expression of phosphorylated protein kinase Akt and eNOS, which are known targets of insulin action.
Retinas from two to three month old Lewis rats were treated with insulin in vitro and the levels of pAkt expression were assessed in lysed tissue homogenates at various time points using an ELISA. Rats were then treated with a single 10 microliter eye drop containing 1% porcine insulin and 1% saponin in a 0.75% sodium bicarbonate buffer in the right eye. At various time points, insulin, pAkt, and eNOS levels were quantified by ELISA.
In vitro, 100 ng/ml of insulin significantly elevated the expression of pAkt above controls at 15 and 30 minutes - fold increases above baseline were 2.3+0.48 and 2.7+0.23, p<0.04 and 0.001, respectively. Similarly, 10 ng/ml of insulin significantly elevated pAkt expression after 15 minutes - fold increase over baseline was 2.2+0.1, p<0.001. In vivo, insulin levels in the retina were elevated to nearly 10 pg/mg protein between 10 and 50 minutes following application. pAkt levels were significantly elevated above baseline controls at 10 and 15 minutes following insulin eye drop application - fold increases above baseline were 3.0+0.25 and 4.5+0.41, p<0.02 and 0.007, respectively. eNOS expression was similarly elevated to 1.3-fold higher than controls (p<0.05) 45 minutes after insulin eye drop application. Experiments to determine the effects of insulin eye drops on NO production are underway.
The above results confirm that insulin accumulates in the rat retina following topical application. Furthermore, our data show that insulin upregulates retinal expression of pAkt and eNOS, mediators that are known to be targets of insulin action. Thus, a long term study designed to determine whether dose-appropriate delivery of insulin to the retina of diabetic rats by topical application can prevent the development of retinopathy appears justified.
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