Abstract
Purpose: :
Evidence for the important role for endoplasmic reticulum (ER) stress in diabetes is emerging, but its contribution to diabetic complications is poorly explored. This study evaluated the role for this phenomenon in diabetic cataract formation and retinal oxidative stress and apoptosis using a pharmacological approach with two chemical chaperones, trimethylamine-N-oxide (TMAO) and 4-phenylbutyric acid (PBA).
Methods: :
Control and STZ-diabetic rats were maintained with or without TMAO (110 mg kg-1d-1, in the drinking water) or PBA (100 mg kg-1d-1, i.p.), for 12 weeks starting from induction of diabetes. Diabetic rats received suboptimal doses of insulin to prevent ketoacidosis and weight loss. Lens clarity was evaluated by indirect ophthalmoscopy and slit lamp examination on weekly basis. Cataracts were scored (0 - clear lenses, 1-vacuoles, 2-opacities, 3-mature cataract). At the end of the study, the rate of apoptosis was assessed by the number of TUNEL-positive nuclei in the flat-mounted retinae. The expression of retinal ER stress variables including total and phosphorylated PKR-like eukaryotic initiation factor 2A kinase (PERK), total and phosphorylated inositol-requiring enzyme-1 (IRE1), and CAAT/enhancer-binding protein homologous protein (CHOP), was measured by Western blot analysis. The indices of oxidative-nitrosative stress, 4-hydroxynonenal (HNE) adducts and nitrotyrosine (NT), were evaluated by ELISA.
Results: :
All the lenses were clear 2 wks after induction of diabetes. During the next 6 wks, TMAO slightly delayed diabetic cataract formation, with significant differences in cataract scores for the 5th wk only (0.667±0.114 vs 0.944±0.151 in untreated diabetic group, p < 0.05). The anticataract effect of PBA was more potent, and was observed till the end of the study, with signifcant differences in the cataract scores for the 10th, 11th, and 12th wks. Rats with 12-wk STZ-diabetes displayed ER stress, manifest by reduced phospho-PERK/PERK ratios, with unchanged phospho-IRE1/IRE1 ratio and CHOP expression. A chemical chaperone treatment increased retinal phospho-PERK/PERK and phospho-IRE1/IRE1 ratios and reduced CHOP expression, thus potentiating unfolded protein response and counteracting ER stress. A 4.3-fold increase in retinal apoptosis in diabetes was completely prevented by TMAO, and essentially prevented by PBA. Both chemical chaperones counteracted diabetes-induced retinal HNE adduct and NT accumulation.
Conclusions: :
ER stress is implicated in diabetes-induced cataract formation, and retinal oxidative-nitrosative stress and apoptosis. The findings identify a new therapeutic target for diabetic ocular complications.
Keywords: diabetic retinopathy • cataract • pathology: experimental