Abstract
Purpose :
In the development of diabetic retinopathy, cytosolic reactive oxygen species (ROS) are elevated before mitochondrial damage can be observed, and NADPH-oxidase 2 (Nox2) and its regulatory cytosolic component, small molecular weight G protein (Rac1), are considered to play a major role. Hyperglycemia is shown to accelerate and exacerbate transcriptional and functional activation of retinal Rac1, and its promoter undergoes active DNA methylation-hydroxymethylation, leading to increased levels of 5hydroxymethyl cytosine (5hmC). Lipid excess also leads to increased ceramide, and serine palmitoyl-transferase (SPT) is the first and the rate-determining enzyme in its biosynthesis. Our aim was to investigate the mechanism by which ceramide promotes Rac1 activation in diabetic retinopathy in type 2 diabetic (T2D) model.
Methods :
C57BL/6 mice were fed high fat diet (45% kcal) for 8 weeks, after which a group of mice were administered a low dose of streptozotocin (30 mg/kg body weight; T2D). Soon after establishment of diabetes, a group of mice received intravitreal administration of 2μg/2μl SPT-siRNA, and these mice were maintained on the same high fat diet for 5 weeks. Age-matched normal mice on a regular mice chow were used as controls. Retinal microvessels (prepared by osmotic shock method) were used to measure Rac1 activity (G-LISA), Nox2 activity (lucigenin) and ROS (DCFDA method). Transcriptional regulation of Rac1 was determined by quantifying 5hmC levels (immuno-capture technique) at its promoter.
Results :
Compared with normal mice, the activity of Rac1, Nox2 and ROS were significantly increased in T2D group, and Rac1 transcripts and 5hmC levels at its promoter were also significantly elevated. Administration of SPT-siRNA prevented increase in Rac1, Nox2, ROS, and also ameliorated 5hmC at Rac1 promoter.
Conclusions :
Thus, accumulation of ceramide in diabetes increases ROS production through transcriptional and functional activation of Rac1. This suggests that regulation of ceramide in diabetic patients with hyperlipidemia may be equally important as regulating their blood sugar to prevent the development of retinopathy.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.