Urolithin C alleviates pancreatic β-cell dysfunction in type 1 diabetes by activating Nrf2 signaling

Abstract

Aims: Type 1 diabetes (T1D) is an autoimmune disorder that destroys insulin-generating pancreatic β-cells. Preserving pancreatic β-cell function is important for treating T1D. Our study aims to explore the mechanism underlying urolithin C (UC)-mediated regulation of β-cell function.

Methods: Non-obese diabetic (NOD) mice were administrated with UC to evaluate UC-mediated protection of T1D. The inflammation of the pancreas islets was examined by hematoxylin and eosin staining. Glucose-stimulated insulin secretion (GSIS) assay and oral glucose tolerance test were applied to evaluate the progression of T1D. MIN6 cells were treated with TNF-α, IL-1β and IFN-γ in the presence of UC. Cell viability was analyzed by CCK-8. Cell apoptosis, proliferation and DNA fragmentation were examined by Annexin V-FITC and PI staining, EdU incorporation and comet assays. Keap1, Nrf2, HO-1 and NQO1 were examined by western blot. Immunofluorescence staining was applied to detect Nrf2 and insulin.

Results: UC administration significantly reduced diabetes incidence, attenuated insulitis, elevated insulin levels and GSIS and reduced blood glucose and AUC in NOD mice. Cytokine treatment suppressed MIN6 cell viability and proliferation but enhanced apoptosis and DNA damage, and these detrimental effects were relieved by UC treatment. Furthermore, UC administration inhibited Keap1 expression and promoted the expression of Nrf2, HO-1 and NQO1 in NOD mice. Nrf2 signaling has been reported to be implicated in preventing the onset of diabetes, and HO-1 and NQO1 are phase II antioxidant enzymes that are regulated by Nrf2 signaling. Cytokine treatment upregulated Keap1 and downregulated Nrf2, HO-1 and NQO1 in MIN6 cells, but it was reversed by UC. The nuclear translocation of Nrf2 was prevented by cytokine treatment, but UC promoted its nuclear translocation. UC-mediated upregulation of Nrf2, HO-1 and NQO1, decreased cell apoptosis and increased proliferation and insulin secretion were abolished by silencing of Nrf2.

Conclusion: UC improves pancreatic β-cell function by activating Nrf2 signaling, thereby alleviating T1D progression.

Fig. 1. UC prevented T1D progression in NOD mice.

A A diagram of animal study design. For early intervention, mice were treated with vehicle or UC (50 mg/kg/day) from 4 weeks of age to 30 weeks of age (sacrifice). Serum was collected at 11 and 19 weeks of age, and insulin levels were examined with ELISA. GSIS and OGTT were determined at 13 and 16 weeks of age. For late intervention, mice were treated with vehicle or UC (50 mg/kg/day) from 10 weeks of age to 26 weeks of age (sacrifice). Serum was collected at 17 and 25 weeks of age, and insulin levels were examined with ELISA. B, C Cumulative diabetes incidence of NOD mice with early and late intervention of UC. Mice treated with vehicle were used as controls (n = 20 for each group). D H&E staining of the pancreas islets from mice (n = 6 for each group. Scale bar: 50 μm). Insulitis was scored 0–4: 0 = clear (no infiltration), 1 = <25% infiltration, 2 = 25~50% infiltration, 3 = 50~75% infiltration and 4 = >75% infiltration. E, F Serum concentrations of insulin were examined by ELISA (n = 10 for each group). G IHC staining of insulin in the pancreas islets (n = 6 for each group. Scale bar: 50 μm). H, I Serum insulin and blood glucose levels were evaluated after GSIS treatment (n = 10 for each group). J Glucose tolerance was evaluated by examining levels of blood glucose at 0, 15, 30, 45 and 60 min and AUC after oral glucose administration (n = 10 for each group). Comparison between the two groups was performed by Student’s t-test. *p < 0.05, **p < 0.01. All experiments were repeated at least three times.

Fig. 2. UC protected pancreatic β-cells against cytokine-induced dysfunction.

A The viability of MIN6 cells treated with UC (0, 2.5, 5, 15, 25, 50 or 100 µM) was analyzed by CCK-8 (n = 3). B MIN6 cells were treated with cytokines in the presence of UC (0, 5, 15 or 25 μM) or verapamil (50 µM). Cell viability was analyzed by CCK-8 (n = 3). In subsequent assays, MIN6 cells were treated with cytokines in the presence of UC (25 μM) or verapamil (50 µM) and divided into four groups: Control, Cytomix, Cytomix + UC and Cytomix + verapamil. C Cell proliferation was analyzed by EdU incorporation (n = 3). D Cell apoptosis was assessed by Annexin V-FITC and PI staining (n = 3). E DNA fragmentation was assessed by comet assays. (n = 3, scale bar: 20 μm). F Glucose-induced insulin secretion was examined by ELISA (n = 3). Comparison among multiple groups was analyzed by ANOVA (followed by Tukey’s post hoc test). *p < 0.05, **p < 0.01 and ***p < 0.001. All experiments were repeated at least three times.

Fig. 3. UC activated Nrf2 signaling in NOD mice.

NOD mice were administrated with UC (50 mg/kg/day) or vehicle from 4 weeks of age. A IF staining of Nrf2 (red) and insulin (green) in the pancreas. Nuclei were stained with DAPI (blue). n = 10. Scale bar: 50 μm. B Protein levels of KEAP1, Nrf2, HO-1 and NQO1 were determined by western blot. β-actin was used as the loading control. n = 10. C Pearson correlation analysis of blood glucose levels and the relative expression of Keap1, Nrf2, HO-1 and NQO1. D Pearson correlation analysis of serum insulin levels and the relative expression of Keap1, Nrf2, HO-1 and NQO1. Comparison between the two groups was performed by Student’s t-test. The correlation of blood glucose or insulin and Keap1, Nrf2, HO-1 and NQO1 was analyzed through the Pearson correlation. *p < 0.05, **p < 0.01. All experiments were repeated at least three times.

Fig. 4. Cytokine treatment-mediated inhibition of Nrf2 signaling was partially relieved by UC.

MIN6 cells were treated with cytokines in the presence of UC and divided into three groups: Control, Cytomix and Cytomix + UC. Nuclear and cytosolic fractions were separated. A Protein levels of KEAP1, nuclear and cytosolic Nrf2, HO-1 and NQO1 were determined by western blot (n = 3). β-actin was used as the loading control. B IF staining of Nrf2 (red). Nuclei were stained with DAPI (blue). Scale bar: 10 μm. Comparison among multiple groups was analyzed by ANOVA (followed by Tukey’s post hoc test). *p < 0.05, **p < 0.01. All experiments were repeated at least three times.

Fig. 5. UC improved cytokine-treated pancreatic β-cell function partly dependent on Nrf2 signaling.

Nrf2 was knocked down in MIN6 cells, and cells were treated with cytokines in the presence of UC and divided into four groups: Control, Cytomix, Cytomix + UC + si-NC and Cytomix + UC + si-Nrf2. A Protein levels of KEAP1, Nrf2, HO-1 and NQO1 were determined by western blot (n = 3). β-actin was used as the loading control. B Cell proliferation was evaluated by CCK-8 (n = 3). C Cell apoptosis was assessed by Annexin V-FITC and PI staining (n = 3). D DNA fragmentation was assessed by comet assays (n = 3, scale bar: 20 μm). E Glucose-induced insulin secretion was examined by ELISA (n = 3). Comparison among multiple groups was analyzed by ANOVA (followed by Tukey’s post hoc test). *p < 0.05, **p < 0.01 and ***p < 0.001. All experiments were repeated at least three times.