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Khasim Syed, Pablo J Marin, Maria E Marin Castano, ; Impaired Akt phosphorylation in response to high insulin in human RPE cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1043.
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© ARVO (1962-2015); The Authors (2016-present)
Chronic hyperinsulinemia is both a marker and a cause for insulin resistance. Insulin, a growth factor essential for retinal cell survival and homeostasis, may play a role in the development of ocular diabetic complications. The effect of a high dose of insulin on the retinal pigment epithelial (RPE) cell, a non-insulin-dependent tissue with an essential role in retinal homeostasis, has received no attention. We will investigate insulin signaling in human RPE cells exposed to hyperinsulinemic environment.
ARPE-19 cells were grown to confluence in modified growth medium, plated at sub-confluent density, and grown to confluence. At the time of confluence, the medium was replaced for 24 hours with phenol red-free medium supplemented with 10% FBS. Cells were then treated in phenol red-free 1% FBS medium with either 500pM or 100nM insulin for different periods of time. Cells were pretreated for 1 hour with 10microM of PI3K inhibitor LY294002 or the proteasome inhibitor MG-132 before they were exposed to high insulin 100nM for 2h. After treatment, cells were harvested for protein extraction or RNA. Expression of insulin receptor, insulin receptor substrate 1 (IRS1), insulin-like growth factor receptors, and Akt were demonstrated by real-time PCR and Western Blot.
We found a robust phosphorylation of Akt at Serine 473 in response to low insulin 500pM, reaching a maximum at 30 minutes before gradually decreasing. These effects are mediated through insulin receptors and not insulin-like growth factor receptors. High insulin 100nM also induced a strong increase in Akt phosphorylation with a peak at 30 minutes followed by a decline. Also, ARPE-19 cells exposed to high insulin 100nM had increased IRS-1 Ser-639 phosphorylation compared to low insulin 500pM. High insulin-induced IRS-1 Ser-639 phosphorylation was blocked by PI3K (LY294002). Exposure to high insulin 100nM for 5 days induced a decline in IRS-1 protein levels with no concomitant change in IRS-1mRNA content. Proteasome-mediated degradation of IRS-1 was involved in this effect because MG-132 abolished high insulin-induced decline in IRS-1 protein expression.
These results suggest that RPE cells sensitivity to insulin may be impaired in hyperinsulemic enivornment through an IRS-1 Ser-639 phosphorylation-dependent mechanism which may promote retinal damage in individuals with persistent high insulin levels.
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