Retinopathy in a Diet-Induced Type 2 Diabetic Rat Model and Role of Epigenetic Modifications

Abstract

Type 2 diabetes accounts for 90% of the population with diabetes, and these patients are generally obese and hyperlipidemic. In addition to hyperglycemia, hyperlipidemia is also closely related with diabetic retinopathy. The aim was to investigate retinopathy in a model closely mimicking the normal progression and metabolic features of the population with type 2 diabetes and elucidate the molecular mechanism. Retinopathy was evaluated in rats fed a 45% kcal as fat diet for 8 weeks before administering streptozotocin, 30 mg/kg body weight (T2D), and compared with age- and duration-matched type 1 diabetic rats (T1D) (60 mg/kg streptozotocin). The role of epigenetic modifications in mitochondrial damage was evaluated in retinal microvasculature. T2D rats were obese and severely hyperlipidemic, with impaired glucose and insulin tolerance compared with age-matched T1D rats. While at 4 months of diabetes, T1D rats had no detectable retinopathy, T2D rats had significant retinopathy, their mitochondrial copy numbers were lower, and mtDNA and Rac1 promoter DNA methylation was exacerbated. At 6 months, retinopathy was comparable in T2D and T1D rats, suggesting that obesity exaggerates hyperglycemia-induced epigenetic modifications, accelerating mitochondrial damage and diabetic retinopathy. Thus, maintenance of good lifestyle and BMI could be beneficial in regulating epigenetic modifications and preventing/retarding retinopathy in patients with diabetes.

Figure 1

Glucose tolerance before and after induction of diabetes. Glucose tolerance was evaluated in overnight-fasted rats maintained on normal chow or high-fat diet before (A) and 2, 4, and 6 months after (B–D) Stz administration. E: Insulin resistance sensitivity was evaluated by measuring glucose in rats after administering Humulin R (1 IU/kg BW). Each graph is representative of 7–9 rats in each group.

Figure 2

Histopathology in retinal microvasculature. Trypsin-digested microvasculature from rats, 4 and 6 months after Stz administration, was stained with PAS, and acellular capillaries (A) and pericyte ghosts (B) were counted. The values in the histograms are means ± SD from 6–10 rats/group each at 4 and 6 months’ duration. C and D: The representative PAS-stained images are from rats at 4 and 6 months’ duration, respectively, and their age-matched controls (Norm and HF, respectively). The arrows indicate acellular capillaries. *P < 0.05 vs. age-matched normal rats.

Figure 3

Functional changes in T2D model. ERG was performed in dark-adapted rats using the Ocuscience HMsERG system. Implicit time (milliseconds [ms]) (A) and amplitude of b-wave at 10,000 mcd.s/m² (B) from 4 months’ to 6 months’ duration are represented as mean ± SD from 5 rats in each group. *P < 0.05 vs. age-matched normal rats.

Figure 4

Mitochondrial damage in retinal microvessels. A: Genomic DNA, isolated from retinal microvessels, was subjected to extended-length PCR using mitochondrial-specific primers for 13.4-kb and 210-bp PCR products. The relative amplification of 13.4-kb and 210-bp products, which is inversely proportional to the mtDNA damage, was calculated. B: Gene transcripts of CytB were quantified by real-time qPCR using β-actin as a housekeeping gene. C: Copy numbers of mitochondria were determined in the genomic DNA by quantifying the ratio of mtDNA-encoded CytB and nuclear DNA-encoded β-actin. Each measurement was made in duplicate in 5–6 rats per group, and the values are represented as means ± SD. Values obtained from normal rats are considered as 100% for mtDNA damage and 1 for CytB mRNA and copy numbers. *P < 0.05 vs. Norm; #P < 0.05 vs. T1D.

Figure 5

Oxidative stress and Rac1 activation: retinal microvessels were used to quantify total ROS fluorometrically using 2′,7′-dichlorofluorescin diacetate (A), Rac1 activation by a G-LISA colorimetric assay kit (B), and Rac1 gene transcripts by qPCR (C) using β-actin as a housekeeping gene. Results are expressed as means ± SD from 6–8 rats in each group, with each measurement made in duplicate. Values obtained from age-matched normal rats are adjusted to 100% for ROS and Rac1 activity and 1 for Rac1 mRNA. *P < 0.05 compared with Norm; #P < 0.05 compared with T1D.

Figure 6

Methylation of mtDNA. Retinal microvessels were used to quantify 5mC levels at mtDNA by methylated DNA immunoprecipitation technique (A) and Dnmt1 gene transcripts by qPCR (B) using β-actin as the housekeeping gene. Values are represented as means ± SD from 4–6 retinal microvessel preparations/group, and the numbers obtained from normal rats are considered as 1. *P < 0.05 vs. Norm; #P < 0.05 vs. T1D.

Figure 7

DNA methylation status of Rac1 promoter in retinal microvessels. A: Hydroxmethylation of Rac1 promoter was evaluated by quantifying 5hmC levels at its promoter using a hydroxyl-methylated DNA immunoprecipitation technique. Binding of Tet2 (B) and the transcription factor (p65 subunit of NF-κB) at Rac1 promoter (C) was determined by chromatin immunoprecipitation technique using IgG as an antibody control. D: Tet2 mRNA was quantified by real-time qPCR, and β-actin was used as a housekeeping gene. E: The enzyme activity of Tets was quantified in 4 months’ duration samples using the TET Activity/Inhibition Assay Kit. The values obtained from normal rats are considered as 1 (100% for Tets activity). *P < 0.05 vs. Norm; #P < 0.05 vs. T1D.