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Bailey Shen, Mariya Ali, Alecia Cutler, Bela Anand-Apte; Insulin Stimulates Increased Production of Soluble Betacellulin in Retinal Pigment Epithelium Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3153.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the effects of high glucose and extracellular insulin on insulin signaling, ADAM 10 (A Disintegrin and Metalloproteinase 10), and betacellulin (BTC) expression in retinal pigment epithelium (RPE) cells in vitro.
ARPE-19 cells were incubated in low (5 mM) or high (25 mM) glucose media containing varying doses of regular human insulin (0 pM - 100 nM). After 48 hours, cells were harvested by mechanical lysis, and western blots of cell lysates were performed to measure expression of insulin receptor, phosphorylated ERK-1 (a member of the insulin signaling pathway), ADAM 10, and betacellulin. As ADAM 10 is known to cleave pro-BTC (MW ~46 kDa) into soluble BTC (22 kDa) and BTC cytoplasmic remnant (~25 kDa), two different types of betacellulin antibodies, one against its N-terminal ectodomain, and one against its cytoplasmic domain, were used to study BTC cleavage.
Insulin concentrations greater than or equal to 10 nM caused increased soluble BTC expression in RPE cells, while glucose concentrations did not have any effect on soluble BTC expression. Insulin also upregulated ADAM 10 expression, but, surprisingly, did not increase levels of BTC cytoplasmic remnant, which would be expected to accumulate if insulin enhanced ADAM 10-mediated cleavage of pro-BTC into soluble BTC. Finally, insulin increased expression of phosphorylated ERK-1 and decreased expression of insulin receptor.
Soluble betacellulin is a molecule known to increase retinal vascular permeability, and a molecule thought to play a role in the pathogenesis of diabetic macular edema. This study suggests that extracellular insulin acting upon RPE cells may contribute to the increased soluble betacellulin previously measured in retinas of diabetic animals and humans. Our results also raise the possibility that insulin-mediated increases in soluble BTC in RPE cells may be independent of pro-BTC cleavage.
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