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Rossella Titone, Meifang Zhu, Ginger Zhou, Danielle M Robertson; Insulin regulates de novo nuclear trafficking of IGF-1/insulin Hybrid-R and metabolism in human corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2622.
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© ARVO (1962-2015); The Authors (2016-present)
The corneal epithelium has been reported as an insulin insensitive tissue that does not require insulin for glucose uptake. In cultured corneal epithelial cells, insulin has been shown to stimulate proliferation and cell migration, although the precise mechanism(s) are not well defined. Our prior work has identified the presence of the IGF-1/insulin hybrid receptor (Hybrid-R) in corneal epithelial cells. The purpose of this study was to investigate the function of insulin in the regulation of Hybrid-R.
Primary cultured (HCEC) and telomerized human corneal epithelial cells (hTCEpi) were cultured in serum-free keratinocyte growth medium (KGM). Prior to stimulation with exogenous insulin, cells were starved in basal medium (KBM) for 24 hours. Western blotting and qPCR were used to determine the expression and subcellular localization of IGF-1R, INSR and Hybrid R. Secretion of IGFBP3, the principal IGF-1 binding protein, was assessed using ELISA. Cell cycle was analyzed using a Cellometer following propidium iodide staining. Metabolic activity was measured in real time using a Seahorse Metabolic Flux Analyzer.
Nutrient starvation significantly upregulated IGF-1R and INSR, driving an increase in Hybrid-R. Accumulating levels of Hybrid-R were detected in the corneal epithelial cell nucleus, which was blocked by treatment with cyclohexamide. Nutrient starvation was associated with cell cycle arrest and a reduction in mitochondrial respiration. The already high level of glycolysis was unchanged. Addition of insulin decreased IGF-1R and INSR to normal levels, restored the cell cycle and mitochondrial function. Similar to hypoxic and hyperglycemic stress, IGFBP3 secretion was significantly increased in response to nutrient deprivation. Unexpectedly, secretion of IGFBP3 was controlled by IGF-1R.
Nutrient deprivation induces upregulation and de novo nuclear trafficking of Hybrid-R. This effect is mediated by insulin and is associated with metabolic changes at the level of the mitochondria. The upregulation of IGFBP3 in response to nutrient starvation suggests that IGFBP3 may function as a stress response protein in corneal epithelial cells through regulation by IGF-1R. Further studies to evaluate the function of IGFBP3 in the corneal epithelium are needed.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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