Abstract
Purpose :
The study of circadian rhythms has been steadily garnering interest with regards to their role in disease development. In addition to the central suprachiasmatic nucleus, many peripheral tissues (i.e retina, liver, and pancreas) are reported to exhibit circadian rhythms of their own. Dysregulation of this process is implicated in disease development, such as diabetes. We tested the hypothesis that pharmacological inhibition of Insulin Receptor Substrate-1 using a specific inhibitor, SecinH3, would alter the retinal circadian rhythm.
Methods :
Freshly enucleated eyes from mPer2Luciferase knockin mice were followed by isolation and culturing of the retina from the eyecup. The retina was gently explanted on a culture membrane with the ganglion cell layer facing up inside a 35-mm culture dish. Each experimental dish was treated with SecinH3 to inhibit IRS-1, while each control dish was treated with corresponding DMSO. Cultures were maintained at 37 degrees C in an incubator, and their bioluminescence was measured continuously in real-time using a Lumicyle® luminometer. For the mRNA extraction, retinas were harvested at allocated times and the RNA was homogenized using Trizol reagent. RNA was converted into a cDNA library using a reverse transcriptase enzyme, and the mRNA expression of clock genes was determined using quantitative PCR.
Results :
Analysis of the phase shift showed the most prominent deviation of the molecular retinal clock in the presence of 100uM SecinH3. Analysis of the period lengths also demonstrated that there was an apparent increase in the periods in the presence of 100uM SecinH3. Gene expression studies indicated there was a significant difference in expression of certain molecular clock gene profiles (i.e Per1 and Cry2)
Conclusions :
Our results are consistent with our hypothesis that inhibition of IRS-1 by SecinH3 alters the circadian rhythm of the retina. Our findings can help understand the role of insulin signaling on circadian rhythms of the retina, and future studies may help to develop novel therapeutics for treating some of the harmful effects of diabetes.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.