Nrf2 and Antioxidant Response in Animal Models of Type 2 Diabetes

Abstract

This perspective examines the proposition that chronically elevated blood glucose levels caused by type 2 diabetes (T2D) harm body tissues by locally generating reactive oxygen species (ROS). A feed-forward scenario is described in which the initial onset of defective beta cell function T2D becomes sustained and causes chronic elevations in blood glucose, which flood metabolic pathways throughout the body, giving rise to abnormally high local levels of ROS. Most cells can defend themselves via a full complement of antioxidant enzymes that are activated by ROS. However, the beta cell itself does not contain catalase or glutathione peroxidases and thereby runs a greater risk of ROS-induced damage. In this review, previously published experiments are revisited to examine the concept that chronic hyperglycemia can lead to oxidative stress in the beta cell, how this relates to the absence of beta cell glutathione peroxidase (GPx) activity, and whether this deficiency might be ameliorated by genetic enrichment of beta cell GPx and by oral antioxidants, including ebselen, a GPx mimetic.

Keywords: Nrf2; antioxidant response; type 2 diabetes in animals.

Figure 1

Metabolic pathways along which glucose metabolism can form reactive oxygen species (ROS). Under physiologic conditions, glucose primarily undergoes glycolysis and oxidative phosphorylation. Under hyperglycemic conditions, excessive glucose levels can swamp the glycolytic process and glyceraldehyde catabolism, so that metabolites are shunted to other pathways, which then generate increasing levels of ROS. Modified from reference [1].

Figure 2

Hypothesis: Under conditions of supraphysiologic concentrations of blood glucose, various metabolic pathways generate increasing levels of ROS in body tissues, including the pancreatic beta cell. The normal beta cell, however, does not contain the full complement of antioxidant enzymes found in other tissues, specifically catalase and glutathione peroxidases, which are important regulators of intracellular ROS levels and catabolism. Consequently, ROS are abundant in beta cells and cause oxidative damage.

Figure 3

Beta cell-specific overexpression of glutathione peroxidase protects beta cells from the functional deterioration observed in wild-type db/db-GPx(−) mice fed a high-fat diet. Initially, blood glucose levels began to rise in both the C57 wild type and the db/db transgenic animals. However, by 10 weeks glucose levels began to decrease to levels lower than those in the transgenic animals, and thereafter returned to the non-hyperglycemic range. We speculate that this delay in glucose response may have been related to the fact that the GPx transgene construct included a glucose-sensitive insulin promoter that was activated by the establishment of hyperglycemia. Modified from Ref. [21].

Figure 4

Comparison of islet morphology in a control (CMC) and an Ebselen-treated ZDF rat. Taken as a whole, the average total beta cell mass in the ebselen group doubled. Bar in left corner of right image indicates relative sizes of both images. Modified from Ref. [22].

Figure 5

(A) Pancreatic sections were double labeled for insulin (green fluorescence) and 4-HNE (a marker for oxidative stress; red fluorescence). ZDF rats after 9 days of a high-fat diet showed intense cytoplasmic staining for 4-HNE in beta cells (B). This was reduced 2 weeks after a return to regular diets (C). Immunostaining for Nrf-2 (red fluorescence) revealed significant immunoreactivity both in the cytoplasm and the nucleus of beta cells ZDF rats fed high-fat diets for 9 days (F) when compared to ZDF controls (E). In contrast, 2 weeks after returning to regular diets the immunoreactivity for Nrf2 was dramatically reduced (G). Similarly, pancreatic sections stained for HO-1 (red fluorescence) showed increased cytoplasmic and nuclear localization of HO-1 (J) in beta cells (green fluorescence), which after 9 days of HFD was greatly diminished 2 weeks after return to regular diet (K). Specificity of detected immunoreactivities was validated by incubation of tissue sections with control IgGs from each species (lower panels). Morphometric analysis of markers of oxidative stress. To determine the presence of oxidative stress, pancreata from both control ZDF rats fed with a 17% fat diet and HFD-treated ZDF rats fed with a 48% fat diet were fixed overnight with 4% formalin. Fixed tissues were processed for paraffin embedding, and 4-μm sections were prepared and mounted on slides. Sections from both groups of animals were chosen at 25-μm intervals for immunolocalization of 4HNE, Nrf2, and HO-1, as well as insulin to identify β cells and E-cadherin to identify the pancreatic epithelium. Fifty sections per animal group were analyzed for morphometric measurements. Primary antibodies used for these experiments included anti-4HNE (Abcam, ab46545), anti-Nrf2 (Abcam, ab31163), and anti–HO-1 (Enzo Life Sciences), all used at 1:100 dilution and incubated overnight at 4 °C. Following reaction with fluorophore-conjugated secondary antibodies (Jackson ImmunoResearch) slides were mounted with DAPI containing mounting medium (Vector Laboratories) and viewed at a Nikon i90 microscope for image acquisition and analysis using NIS-Elements AR 3.2 (Nikon). *** p < 0.001, **** p < 0.0001. Modified from Ref. [23].

Figure 6

Illustration of the functional relationships between KEAP1 and Nrf2 under conditions of non-stress and oxidative stress. Under quiescent conditions, Nrf2 is bound by KEAP1 in the cytoplasm, which ushers Nrf2 to proteosomes for degradation. In the face of oxidative stress, Nrf2 is released from KEAP1 and enters the nucleus, where it serves as a key activator for the promoter of antioxidant genes.