Abstract
Abstract: :
Purpose:Mounting evidence indicates that insulin can act as a neuroprotective factor in the central nervous system, including the retina. However, a physiological role of insulin in retinal neurons is unknown. The purpose of this study is to test whether insulin regulates expression of specific genes in retinal neurons. Methods:R28cells were differentiated to neurons and treated with 10 nM insulin for varying periods with or without 5 micro M LY294002 or 25micro M PD098059. RNA was extracted and reverse-transcribed with 32P labeling. Radiolabeled cDNA was hybridized to membranes on which more than 1000 cDNA fragments of known genes were spotted. After stringent washes, the membranes were subjected to phosphoimager analysis. For the genes whose expression was either upregulated or downregulated by 2-fold or more, Northern blotting was performed to verify the differential expression. Similar experiments were conducted with IGF-1. Results:Microarray experiments demonstrated that incubation of R28 cells with insulin for 24 hours increased mRNA transcripts of 7 known genes 2-fold or more, whereas insulin decreased mRNA transcripts of 2 genes. Among those, Northern blotting confirmed a significant increase in the amounts of Na+/K+ ATPase alpha 1 subunit and Nur 77 mRNA transcripts in a time-dependent fashion. The former maximally increased at 6 hours after insulin stimulation, while the latter did at 1 hour. Co-treatment of the cells with LY294002, a PI3K inhibitor, blocked the effect of insulin to increase the Na+/K+ ATPase alpha 1 subunit transcript. However, PD098059 did not affect the insulin-mediated increase in the gene transcript. IGF-1 also increased the amount of the gene transcript, that effect was prolonged up to 24 hours after stimulation. In addition, 10 nM insulin injected intravitreally in normal rats increased retinal Na+/K+ ATPase alpha 1 subunit mRNA content 2-fold between 6 and 24 hrs. Conclusion:Insulin regulates expression of Na+/K+ ATPase alpha 1 subunit gene in a PI3K pathway-dependent fashion in retinal neurons. Insulin may play a fundamental role in the neurosensory retina to maintain its function.
Keywords: 482 NaK ATPase • 580 signal transduction • 554 retina