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X Wu, C Reiter, DA Antonetti, TW Gardner; Insulin Stimulates Cell Survival in a p70S6 Kinase-Dependent Manner in Retinal Neurons . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1336.
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Purpose: Previously we showed that insulin rescues retinal neurons from apoptosis by a PI-3-K/Akt mediated mechanism. Akt is a key factor in the insulin's anti-apoptotic effects. This study examines insulin signaling distal to Akt. Methods: R28 retinal neuronal cells were cultured in laminin-coated plates for 24h and then exposed to different treatment. For cell signaling studies, cells were changed to regular medium with 5mM glucose (LG) or medium with 20mM glucose (HG) or with 1.5mM glucosamine (GN) for another 24h, followed by 2h serum starvation and insulin (10nM) treatment. Cell lysates were immunoblotted with antibodies for phosphorylated and total Akt, p70S6 Kinase, and FKHR. For apoptosis studies, R28 cells were cultured in serum free medium with LG, HG or GN for additional 24h with or without insulin or rapamycin (10nM) or Ro-31-8220 (10nM). Hoechst staining was carried out and pyknotic cells were counted as a percent total cells. Results: Insulin phosphorylated Akt, p70S6K and FKHR within 2min and HG and GN decreased these effects. LY294002 blocked Akt, p70S6K and FKHR phosphorylation. Rapamycin blocked p70S6K phosphorylation but not Akt or FKHR. Insulin decreased the number of pyknotic R28 cells, whereas HG and GN increased the number of pyknotic R28 cells as determined by Hoechst staining. When pretreated with rapamycin or Ro-31-8220 before insulin treatment, the number of pyknotic cells was increased 3-fold versus insulin alone. Neither rapamycin nor Ro-31-8220 affected pyknotic cell numbers cultured with serum. Conclusion: Insulin affects multiple factors downstream of PI-3-K/Akt in retinal neurons. These findings may have importance for the understanding of diabetic retinopathy.
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