Nrf2 represses the onset of type 1 diabetes in non-obese diabetic mice

in Journal of Endocrinology
Correspondence should be addressed to M Yamamoto or A Uruno: masiyamamoto@med.tohoku.ac.jp or uruno@med.tohoku.ac.jp
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The transcription factor Nrf2 (NF-E2-related factor 2) plays a critical role in oxidative stress responses. Although activation of Nrf2 signaling is known to exert anti-inflammatory effects, the function of Nrf2 in inflammation-mediated autoimmune disorders, such as type 1 diabetes, is not well established. To address the roles of Nrf2 in protection against autoreactive T-cell-induced type 1 diabetes, we used non-obese diabetic (NOD) mice, which are a polygenic model of human type 1 diabetes, to generate a genetic model for assessment of the contribution of Nrf2 activation to prevention and/or treatment of type 1 diabetes. Because Keap1 (Kelch-like ECH-associated protein 1) negatively regulates Nrf2, we used Keap1 gene knockdown driven by either hypomorphic or knockout Keap1 alleles, which enhanced Nrf2 signaling to moderate or excess levels, respectively. Nrf2 activation in the NOD::Keap1 FA/ mice inhibited T-cell infiltration within or near the islets, ameliorated impairment of insulin secretion and prevented the development of diabetes mellitus. Notably, Nrf2 activation decreased both the plasma interferon-γ (IFN-γ) levels and the IFN-γ-positive cell numbers in the pancreatic islets. The amelioration of diabetes was also observed in the NOD mice with two hypomorphic Keap1 alleles (Keap1FA/FA) by intermediate activation of Nrf2. Both NOD::Keap1FA/ and NOD::Keap1FA/FA mice had a decreased incidence of diabetes mellitus, demonstrating that activation of Nrf2 signaling prevented the onset of type 1 diabetes mellitus in NOD mice. Thus, Nrf2 appears to be a potential target for the prevention and treatment of type 1 diabetes.

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  • Supplementary Figure 1. Correlation between blood glucose levels and Ifng mRNA expression levels of spleen in the NOD::Keap1FA/+ and NOD::Keap1FA/– mice at 24 weeks of age (n=13 in each group) . The mRNA expression data were normalized to Hprt, and the expression levels in hyperglycemic NOD::Keap1FA/+ mice were set as 1.
  • Supplementary Table 1

 

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    A mouse model of type 1 diabetes with concomitant activation of Nrf2 signaling activation. (A) Keap1FA/+ mice were backcrossed with NOD/Shi background mice for eight generations (N8). The mice were mated to obtain NOD::Keap1+/+, NOD::Keap1FA/+ and NOD::Keap1FA/FA genotype mice. The NOD::Keap1+/+ and NOD::Keap1FA/FA mice were examined for eight successive generations (N8) as indicated. (B) Nqo1 mRNA expression levels in the spleens of the NOD::Keap1+/+ (n = 5), NOD::Keap1FA/+ (n = 4) and NOD::Keap1FA/FA mice (n = 6). The data were normalized to β-actin, and the expression levels in the NOD::Keap1+/+ mice were set as 1. (C) The Keap1FA/FA and Keap1+/ mice were backcrossed with NOD/Shi background mice for 12 and 11 generations (N12 and N11), respectively. The NOD::Keap1FA/FA mice and NOD::Keap1+/ mice were mated, and NOD::Keap1FA/ and NOD::Keap1FA/+ mice were generated as indicated. (D) Nqo1 mRNA expression levels in the spleens of the NOD::Keap1FA/+ (n = 14) and NOD::Keap1FA/ (n = 13) mice. The data were normalized to β-actin, and the expression levels in the NOD::Keap1FA/+ mice were set as 1. The bar represents the median. ANOVA followed by Fisher’s LSD post hoc test for (B) and Student’s t-test for (D) were applied. *P < 0.05, **P < 0.01 and ***P < 0.001.

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    Activation of Nrf2 signaling decreases the incidence of diabetes in NOD mice. (A) Kaplan–Meier survival curves showing the percentages of the NOD::Keap1FA/+ (control, n = 7) and NOD::Keap1FA/ (Keap1 knockdown, n = 11) mice that were diabetes-free up to 24 weeks of age. Diabetes was defined as a blood glucose level >250 mg/dL after 2 consecutive biweekly measurements. (B and C) Changes in blood glucose levels (B) and body weights (C) in the NOD::Keap1FA/+ (n = 7) and NOD::Keap1FA/ (n = 11) mice. The data are the mean ± s.e.m. (D) Plasma insulin levels in the NOD::Keap1FA/+ mice (n = 7) and NOD::Keap1FA/ (n = 11) at 24 weeks of age. The bar represents the median. (E) Correlation between the plasma insulin levels and blood glucose levels in the NOD::Keap1FA/+ (n = 7) and NOD::Keap1FA/ (n = 11) mice at 24 weeks of age. Note that high blood glucose levels (i.e., diabetic) were concentrated in the NOD::Keap1FA/+ genotype, whereas none of the NOD::Keap1FA/ mice had diabetes. Log-rank tests for the Kaplan–Meier survival curves for (A), the repeated-measures ANOVA followed by Fisher’s LSD post hoc test for (B and C) and Student’s t-test for (D) were performed. P < 0.01. *P < 0.05, **P < 0.01 and ***P < 0.001 vs the NOD::Keap1FA/+ mice.

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    Activation of Nrf2 signaling prevents insulitis in NOD mice. (A) Representative images of pancreatic islets from hyperglycemic (blood glucose >400 mg/dL) and normoglycemic (blood glucose <150 mg/dL) NOD::Keap1FA/+ mice (left and middle panels, respectively) and normoglycemic NOD::Keap1FA/ mice (right panel) at 24 weeks of age. Bar, 100 μm. Arrowhead, extensive inflammatory cell infiltration in the islets. (B) Percentage of islets with insulitis in the NOD mice. Nine to fifteen islets of each mouse were examined for percentage of insulitis in the NOD::Keap1FA/+ (n = 10) and NOD::Keap1FA/ (n = 5) mice, respectively. The mean value of individual mice is shown. The bar represents the median. (C) Distribution of the insulitis scores for the NOD mice. The percentages of islets with each insulitis grade were calculated using pooled values from 70 to 130 islets from the NOD::Keap1FA/+ (n = 10) and NOD::Keap1FA/ (n = 5) mice. (D) Total insulitis scores for individual NOD mice. Nine to fifteen islets in each mouse were examined, and the 5-graded scoring scale (0, 1, 2, 3 and 4) multiplied by the islet number for each grade was summed up for each individual mouse. The mean value is shown in NOD::Keap1FA/+ (n = 10) and NOD::Keap1FA/ (n = 5) mice. The bar represents the median. Scoring scale: grade 0, no infiltration; grade 1, predominantly peri-islet mononuclear cell infiltration; grade 2, infiltration in 25–50% of the islets; grade 3, infiltration in 50–75% of the islets; and grade 4, infiltration in >75% of the islets. Mann–Whitney U test (B) and Student’s t-test (D) were applied. No statistical analysis was performed in (C). *P < 0.05.

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    Activation of Nrf2 signaling protects insulin-positive cells in NOD mice. (A) Immunofluorescent staining for insulin (green) and glucagon (red) in islets from the hyperglycemic and normoglycemic NOD::Keap1FA/+ mice and the normoglycemic NOD::Keap1FA/ mice at 24 weeks of age (nuclei are stained with DAPI, blue). Bar, 100 μm. (B and D) Insulin-positive (B) and glucagon-positive (D) areas in the whole pancreatic sections. The diabetic (DM) and non-diabetic (non-DM) NOD::Keap1FA/+ mice and the non-DM NOD::Keap1FA/ (n = 5 in each group) mice were examined at 24 weeks of age. The bar represents the median. (C, E and F) Pancreatic insulin (C) and glucagon (E) contents and plasma glucagon levels (F) in the DM and non-DM NOD::Keap1FA/+ mice and the non-DM NOD::Keap1FA/ mice (n = 5, 9 and 6, respectively) at 15 weeks of age. The bar represents the median. (G) Immunohistochemistry for NQO1 in pancreatic sections from the NOD::Keap1FA/ and NOD::Keap1FA/+ mice. Bar, 100 μm. ANOVA followed by Fisher’s LSD post hoc test was applied. **P < 0.01 and ***P < 0.001.

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    Inflammation-related gene expression levels in the spleens of the NOD mice. (A, B and C) mRNA expression levels of the inflammation-related genes Ifng (A), Tnf (B) and Cd68 (C) in the spleens of the DM and non-DM NOD::Keap1FA/+ mice and the non-DM NOD::Keap1FA/ mice (n = 4, 10 and 13, respectively) at 24 weeks of age. The data were normalized to Hprt for Ifng and to β-actin for Tnf and Cd68, and the expression levels in the DM NOD::Keap1FA/+ mice were set as 1. The bar represents the median. ANOVA followed by Fisher’s LSD post hoc test was applied. The Grubbs’ test revealed that one non-DM NOD::Keap1FA/+ sample was an outlier in Ifng mRNA expression, which was removed from the data set, showing 9 points in (A). A sample of non-DM NOD::Keap1FA/ did not show any signal of Tnf mRNA expression, which was removed from the data set, then 12 points were displayed in (B). **P < 0.01 and ***P < 0.001.

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    Islet-infiltrating T-cells in Nrf2-activated NOD mice. (A) Plasma IFN-γ levels in the DM and non-DM NOD::Keap1FA/+ mice and the non-DM NOD::Keap1FA/ (n = 5, 10 and 13, respectively) mice at 24 weeks of age. The data are the mean ± s.e.m. (B) Immunohistochemistry for IFN-γ in pancreatic sections from the NOD::Keap1FA/ and NOD::Keap1FA/+ mice. The dotted lines indicate islets, the black lines indicate infiltrated lymphocytes and the arrowheads indicate IFN-γ-positive cells. Bar, 100 μm. (C) Numbers of IFN-γ-positive cells per islet in the NOD mice. The individual mouse values for IFN-γ-positive cell number per islet were examined. The mean value is shown in NOD::Keap1FA/+ (n = 6) and NOD::Keap1FA/ (n = 5) mice. The bar represents the median. (D) Immunohistochemistry for Foxp3 in pancreatic sections from the NOD::Keap1FA/ and NOD::Keap1FA/+ mice. The dotted lines indicate islets, the black lines indicate infiltrated lymphocytes and the arrowheads indicate Foxp3-positive cells. Bar, 100 μm. (E) The percentages of islets with Foxp3-positive cells in the NOD mice. The percentage of islets showing Foxp3-positive cells in the islets and surrounding areas were counted for individual mice. The mean value is shown in the NOD::Keap1FA/+ (n = 8) and NOD::Keap1FA/ (n = 5) mice. The bar represents the median. ANOVA followed by Fisher’s LSD post hoc test (A) and Mann–Whitney U test (C and E) were applied. *P < 0.05.

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    Activation of Nrf2 decreases the plasma β-hydroxybutyrate levels in NOD mice. (A) The plasma β-hydroxybutyrate levels in the NOD::Keap1FA/+ (n = 10) and NOD::Keap1FA/ (n = 8) mice at 24 weeks of age. The bar represents the median. (B) Correlation between the plasma insulin and β-hydroxybutyrate levels in the NOD::Keap1FA/ and NOD::Keap1FA/+ mice at 24 weeks of age. (C, D and E) The mRNA expression levels of the β-hydroxybutyrate synthesis-related genes Hadh (C), Bdh1 (D) and Hmgcs2 (E) in the livers of the NOD::Keap1FA/+ (n = 5) and NOD::Keap1FA/ (n = 11) mice at 24 weeks of age. The data were normalized to Hprt, and the expression levels in the controls were set as 1. (F) Nqo1 mRNA expression levels in the livers of the NOD::Keap1FA/+ (n = 5) and NOD::Keap1FA/ (n = 11) mice at 24 weeks of age. The data were normalized to β-actin, and the expression level in the controls was set as 1. The bar represents the median. Student’s t-test was applied. *P < 0.05 and **P < 0.01.

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    An intermediate level of Nrf2 activation in NOD mice also shows protection. (A) Kaplan–Meier survival curves showing the percentage of diabetes-free NOD::Keap1+/+ (n = 11) and NOD::Keap1FA/FA (n = 12) mice. Diabetes was defined as a blood glucose level >250 mg/dL after consecutive biweekly measurements. (B and C) Blood glucose levels (B) and body weights (C) in the NOD::Keap1+/+ (n = 11) and NOD::Keap1FA/FA (n = 10) mice at 22 weeks of age. The bar represents the median. Log-rank tests for the Kaplan–Meier survival curves (A) and Student’s t-test (B and C) were performed. *P < 0.05.

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