LXR Activation May Correct Glucose Mediated Dysfunction In Vitro and Modulate Diabetic Retinopathy (DR) In Type 1 Diabetes Model

Abstract

Purpose: : Diabetes is associated with hypercholesterolemia as well as hyperglycemia which can lead to DR. Liver X receptor (LXR) not only modulates cholesterol but also reduces inflammation and activates the protective arm of the renin-angiotensin system (RAS) through activation of ACE 2, Ang 1-7 and its receptor Mas1. We postulated that LXR activation may serve to correct glucose mediated dysfunction in vitro and improve diabetic retinopathy.

Methods: : 661W murine cone cells and ARPE-19 human RPE cells were cultured in media either without (control) or with 5mM and 30 mM Glucose (G-5 and G-30). On the 7^th day of glucose treatment, 1µM of LXR agonist-GW3965 hydrochloride (GW) was added to cultures containing glucose (G-5+GW and G-30+GW). On the following day cells were harvested and protein and mRNA expression for LXR α/β, RAS related genes, and iNOS were determined by western blot and quantitative PCR, respectively (n=4). In parallel studies DBA/2J mice made diabetic with streptozotocin were treated with diet containing the LXR agonist GW3965 (10 mg/kg/day) or vehicle for 12 weeks and isolated retinas were processed for trypsin digestion.

Results: : Western blot analysis demonstrated that LXR-β expression was reduced in the high glucose treated group (G-30) (p<0.05) which was corrected by GW3965 (p<0.01) treatment in cone cells. In contrast, LXR protein could not be detected in ARPE-19 cells under any of the experimental conditions tested. iNOS protein expression was increased in G-30 group (p<0.001) compared to control which was reversed by GW3965 (p<0.001); whereas Mas-1 protein expression was reduced in diabetic group (G-30) (p<0.05) which was corrected by GW3965 (p<0.001) in both cell types. iNOS mRNA expression was increased 3 and 6 fold in cone cells and ARPE-19 cells respectively in the G-30 group which could be reversed by GW3965; mRNA expression of Mas1 was decreased 5-fold by high glucose (G-30) group compared to control and this was reversed by GW3965 in ARPE-19 cells. Quantification of trypsin digested retinas revealed a 50% reduction in the number of acellular capillaries (p<0.05) in the group treated with GW3965 as compared to vehicle treated mice.

Conclusions: : Liver X Receptor (LXR) agonists can correct glucose-induced changes in iNOS and increase expression of Mas1 to activate the protective arm of the RAS and may represent a novel therapeutic target for DR.

Keywords: diabetic retinopathy • oxidation/oxidative or free radical damage • retinal culture