Abstract
Purpose :
Diabetic retinopathy (DR) is a growing health concern with limited treatment options and is the leading cause of blindness in working age adults. Previous research has demonstrated dyslipidemia and dysregulation of cholesterol levels play a significant role in DR development; however, strategies to normalize cholesterol regulation in the retina are limited. Major regulators of cholesterol metabolism are Liver X-receptors-α/β (LXR). LXR signaling has been shown to activate reverse cholesterol transport (RCT). SIRT1 is a nutrient-sensing deacetylase activated during fasting, and a major regulator of LXR activity. Both SIRT1 and LXR levels have been shown to be decreased in diabetic retina. Thus, we hypothesize that the fasting-induced increase in SIRT1 followed by LXR deacetylation and activation leads to an increase in RCT and lowering of cellular cholesterol levels.
Methods :
Bovine retinal endothelial cells (BRECs) were isolated and validated according to a previously published protocol. BRECs were treated with diabetic relevant stimuli TNFα (10ng/ml) for 24hrs. In order to model calorie restriction in vitro BRECs were serum starved (0% FBS) for 24hrs. ABCA1 and ABCG1 were analyzed by qRT-PCR. Cellular cholesterol concentrations were measured using the Amplex Red Cholesterol Assay Kit. Cell viability was measured via trypan blue exclusion assay.
Results :
Serum starvation increased SIRT1 mRNA levels (n=3; p=0.001), as well as LXR-controlled genes ABCA1 (n=3; p<0.01), and ABCG1 (n=3; p<0.01) mRNA levels. Additionally, treatment with TNFα increased cholesterol levels (n=6, p=.0233) while serum deprivation for 24hrs caused decreased cholesterol levels in BRECs (2% FBS; p=0.0025, 0% FBS; p<0.001, n=6). There was no significant difference in cell viability among cells cultured in 10% FBS, 2% FBS or 0% FBS after 24hrs.
Conclusions :
The results of this study show that serum starvation promotes activation of the SIRT1-LXR pathway. Activation of this signaling pathway, in turn, promotes cholesterol metabolism in retinal endothelial cells. Since it has been previously shown that dyslipidemia plays a central role in progression of DR, activation of pathways that promote cholesterol efflux and metabolism may represent novel therapeutic targets for treatment of DR.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.