Antioxidants Complement the Requirement for Protein Chaperone Function to Maintain β-Cell Function and Glucose Homeostasis

Abstract

Proinsulin misfolding in the endoplasmic reticulum (ER) initiates a cell death response, although the mechanism(s) remains unknown. To provide insight into how protein misfolding may cause β-cell failure, we analyzed mice with the deletion of P58(IPK)/DnajC3, an ER luminal co-chaperone. P58(IPK-/-) mice become diabetic as a result of decreased β-cell function and mass accompanied by induction of oxidative stress and cell death. Treatment with a chemical chaperone, as well as deletion of Chop, improved β-cell function and ameliorated the diabetic phenotype in P58(IPK-/-) mice, suggesting P58(IPK) deletion causes β-cell death through ER stress. Significantly, a diet of chow supplemented with antioxidant dramatically and rapidly restored β-cell function in P58(IPK-/-) mice and corrected abnormal localization of MafA, a critical transcription factor for β-cell function. Antioxidant feeding also preserved β-cell function in Akita mice that express mutant misfolded proinsulin. Therefore defective protein folding in the β-cell causes oxidative stress as an essential proximal signal required for apoptosis in response to ER stress. Remarkably, these findings demonstrate that antioxidant feeding restores cell function upon deletion of an ER molecular chaperone. Therefore antioxidant or chemical chaperone treatment may be a promising therapeutic approach for type 2 diabetes.

Figure 1

Deletion of P58^IPK in mice causes β-cell failure. A: Blood glucose was measured in P58^IPK+/+ and P58^IPK−/− mice at the indicated age after a 5-h fast (n = 5 or 6 mice per group). B: A GTT was performed (n = 5 or 6 mice [6–7 weeks of age] per group). C: An insulin tolerance test was performed (n = 5 or 6 mice [9–10 weeks of age] per group). D: Serum insulin after a 5-h fast was measured in 16- to 20-week-old mice after a 5-h fast. E: Western blot analysis was performed using 100 islets isolated from 10-week-old mice. F: Pancreatic insulin content was measured and normalized to protein content (n = 5 mice [10–12 weeks of age] per group). G: Islet mass was measured in pancreatic sections from 16- to 20-week-old mice. The percentage indicates the total islet mass over the pancreas area in each pancreata (n = 3 mice per group). H: Immunohistochemistry for insulin and glucagon was performed on pancreatic sections from 10- to 12-week-old mice. Representative images are shown. Scale bar represents 100 μm. I: Transmission electron microscopy was performed on pancreatic sections from 12-week-old mice. Representative images are shown. Scale bar represents 2 μm in upper panels and 0.5 μm in lower panels. ER, rough endoplasmic reticulum; M, mitochondria. *P < 0.05; **P < 0.01.

Figure 2

Deletion of P58^IPK causes oxidative stress in β-cells. A: Immunohistochemistry for nitrotyrosine was performed on pancreatic sections from 12-week-old mice. Representative images are shown. Scale bar represents 100 μm. B: HODEs were measured in extracts from 50 islets isolated from 10- to 12-week-old mice (n = 6 mice per group). C: Quantitative RT-PCR was performed using total RNA extracted from islets from P58^IPK+/+ and P58^IPK−/− mice at 4, 6–7, or 10 weeks of age (n = 3–6 mice per group). All data are mean ± SEM. *P < 0.05; **P < 0.01.

Figure 3

P58^IPK−/− β-cells are more susceptible to ER stress. A–C: Quantitative RT-PCR was performed using total RNA extracted from P58^IPK+/+ and P58^IPK−/− β-cell lines treated with Tm (2 μg/mL) for 0, 8, or 24 h: pancreatic β-cell-specific genes (A); cell death genes (B); and UPR genes (C). D: Cell viability was measured in P58^IPK+/+ and P58^IPK−/− β-cell lines treated with vehicle (Veh) (DMSO), Tm (2 μg/mL), or thapsigargin (Tg; 300 nmol/L) for 24 h and 0.6 mmol/L H₂O₂ for 8 h. E: Western blot analysis was performed using P58^IPK+/+ and P58^IPK−/− β-cell lines treated with vehicle (DMSO), Tg (300 nmol/L), and Tm (2 μg/mL) for 24 h. F: Western blot analysis was performed using P58^IPK+/+ and P58^IPK−/− β-cell lines treated with Tm (2 μg/mL) for 24 h. Cell lysates were harvested at the indicated time points. G and H: P58^IPK+/+ and P58^IPK−/− β-cell lines were infected with adenovirus-expressing β-gal (rAd-β-gal) or Akita proinsulin (rAd-Akita) for 24 h. G: Total RNA was isolated for quantitative RT-PCR. H: Cell viability was measured. All data are mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01. hr, hours.

Figure 4

Chemical chaperone treatment preserves β-cell function in P58^IPK−/− mice. P58^IPK+/+ and P58^IPK−/− mice aged 22–26 weeks were treated intraperitoneally with saline or PBA (500 mg/kg body weight/day) for the indicated durations. Blood glucose was measured after a 5-h fast (A) and GTTs were performed before and after treatment with PBA for 42 days (B) (n = 4 or 5 mice per group). Significant differences between P58^IPK−/− mice treated with saline or PBA are shown. *P < 0.05; **P < 0.01. Significance was shown between P58^IPK−/− and P58^IPK−/− with PBA treatment.

Figure 5

Chop deletion preserves β-cell function in P58^IPK−/− mice. A: Blood glucose was measured at the indicated times, either ad libitum or after a 5-h fast, in mice with the indicated genotypes up to 41 weeks of age (n = 7–13 mice per group). B: GTTs were performed in mice with the indicated genotypes. Glucose doses were 2 mg/kg body weight for mice 14 and 16 weeks of age and 1 mg/kg body weight for mice 36 weeks of age (n = 7–13 mice per group). Significance was shown between P58^IPK−/− Chop^+/− and P58^IPK−/− Chop^−/−. C: Pancreatic insulin was measured in mice with the indicated genotypes at 16 weeks of age (n = 4–6 mice per group). D: Islet mass was measured in pancreatic sections from 16-week-old P58^IPK−/− Chop^+/− or P58^IPK−/− Chop^−/− mice and their littermates. The percentage indicates the total islet mass over the pancreas area in each pancreata (n = 4 mice per group). E: Transmission electron microscopy was performed on pancreatic sections from P58^IPK+/−Chop^+/− mice and P58^IPK−/−Chop^−/− mice at 38–41 weeks of age. Representative images are shown. Scale bar represents 0.5 μm. All data are mean ± SEM. *P < 0.05; **P < 0.01.

Figure 6

Antioxidant treatment prevents β-cell failure in P58^IPK−/− mice. A and B: Mice with the indicated genotypes at 20–22 weeks of age were fed control chow or chow supplemented with BHA for up to 39 weeks (n = 3 or 4 mice per group). A: Blood glucose was measured either ad libitum or after a 5-h short fast. B: GTTs were performed on mice with the indicated genotypes before and 2 and 40 weeks after feeding with control or BHA-supplemented chow (n = 5–7 mice per group). Significance was shown between P58^IPK−/− mice fed with control or BHA-supplemented chow. C: Pancreatic insulin contents were measured in mice fed control or BHA-supplemented chow for 40 weeks (n = 5 or 6 mice per group). D: Islet mass was measured in pancreatic sections from 16- to 20-week-old P58^IPK−/− mice fed control or BHA-supplemented chow for 6 weeks. The percentage indicates the total islet mass over the pancreas area in each pancreata (n = 4 mice per group). E: GSIS (left panel) and insulin contents in total islet lysates (right panel) were measured in mice with the indicated genotypes fed control chow or BHA-supplemented chow for 40 weeks. Islets from two animals per condition were analyzed in triplicate. F: Insulin and proinsulin immunohistochemistry was performed on pancreatic sections from P58^IPK+/+ and P58^IPK−/− mice fed control or BHA-supplemented chow for 40 weeks. Representative images are shown. Scale bars represent 30 μm. G: Transmission electron microscopy was performed on pancreatic sections from P58^IPK−/− mice fed control or BHA-supplemented chow for 22 weeks. Representative images are shown. Scale bar represents 0.5 μm. *P < 0.05; **P < 0.01.

Figure 7

Antioxidant feeding improves β-cell function in P58^IPK−/− mice. A: HODEs were measured in extracts from 50 islets isolated from mice aged 7–9 weeks that were fed control chow or BHA-supplemented chow for 3 weeks (n = 5–7 mice per group). B: Immunohistochemistry for 4-HNE was performed on pancreatic sections from P58^IPK−/− mice fed control chow or BHA-supplemented chow for 6 weeks. Representative images are shown. Arrows indicate nuclei. Scale bar represents 100 μm. C: Immunohistochemistry for MafA and insulin was performed on pancreatic sections from P58^IPK+/+ or P58^IPK−/− mice fed control or BHA-supplemented chow for 6 weeks. Representative images are shown. Scale bar represents 100 μm. All data are mean ± SEM. **P < 0.01.

Figure 8

BHA feeding improves glucose homeostasis in Akita mice. Wild-type and heterozygous Akita+ male littermate mice 10–20 weeks of age were fed control chow or BHA-supplemented chow for up to 36 weeks. A: Blood glucose concentrations were measured between 9 and 10 a.m. ad libitum (n = 3 or 4 mice per group). B: GTTs were performed before and 8 weeks after feeding control chow or BHA-supplemented chow. Glucose (1 g/kg body weight) was administered intraperitoneally to mice fasted for 5–6 h (n = 3 or 4 mice per group). C: Model depicting β-cell demise caused by ER protein misfolding, subsequent ROS production, and interventions demonstrated to preserve β-cell function. All data are mean ± SEM. **P < 0.01.