Fig 1.
MLDS treatment induces a diabetic phenotype in the absence of DJ-1.
Male control and DJ-1 KO mice at 12–13 weeks of age were treated with 40 mg STZ/kg body weight on five consecutive days. Blood glucose concentrations, glucose tolerance, and plasma insulin concentrations were determined. (a,c) Random (a) and fasting (c) blood glucose concentrations in control (black squares) and DJ-1 KO mice (grey squares). n = 6–8 mice per experimental group. (b,d) Corresponding areas under the curve (AUC) to (a,c) are shown for control (black columns) and DJ-1 KO (grey columns) mice each. (e,f) Glucose tolerance test (e) and its corresponding AUC (f) in 14–16 weeks-old control (black squares and black column) and DJ-1 KO mice (grey squares and grey column). The glucose tolerance test was performed after intraperitoneal administration of glucose (1 g/kg body weight). n = 8 mice per experimental group. (g,h) Relative fasting (g) and non-fasting (h) plasma insulin concentrations in 14–16 weeks-old control (black columns) and DJ-1 KO mice (grey columns) normalised to controls. n = 8 mice per experimental group in (g) and n = 5 in (h). *p<0.05 (Student’s t-test in b, d, f-h. Student’s t-test with Holm-Bonferroni correction in a, c, e). All values are means ± SD.
Fig 2.
DJ-1 is required to lower the rate of apoptosis upon MLDS treatment.
(a-h) LSM images of pancreatic islets in sections of pancreata from male control (a-d) and DJ-1 KO mice (e-h) stained for cell nuclei (DAPI) (a,e), insulin (b,f) and TUNEL positive nuclei (c,g). Merged images (d,h) are also shown. Scale bar, 25 μm. (i) Quantification of the relative amount of TUNEL positive nuclei in pancreatic islets of DJ-1 KO and control mice, calculated as total number of TUNEL positive apoptotic nuclei per insulin positive area expressed as percentage of control. n = 3 pancreata per experimental group. *p<0.05 (Student’s t-test). Data are expressed as means ± SD.
Fig 3.
DJ-1 is required to reduce loss of beta cell mass following treatment with MLDS.
(a,d) Representative fluorescence microscopy images of pancreatic sections from 16 weeks-old male control and DJ-1 KO mice following MLDS treatment, stained for cell nuclei (DAPI) and beta cells (insulin). Scale bars, 0.5 mm. (b,c,e,f) LSM images of pancreatic islets in sections of pancreata from male control (b,c) and DJ-1 KO mice (e-f) after MLDS treatment, stained for cell nuclei (DAPI) and beta cells (insulin) (b,e). Merged images (c,f) are also shown. Scale bars, 50 μm. (g) Morphometric analyses of relative beta cell area from DJ-1 KO and control mice calculated as insulin-positive area per total nuclei area of evenly spaced pancreatic sections. n = 3 mouse pancreata per experimental group. Beta cell area was quantified after four weeks of STZ treatment. For comparison, untreated control/wild type mice without STZ treatment were included. *p<0.05 (One-way ANOVA followed by Tukey´s multiple comparison test). Data are expressed as means ± S.D.
Fig 4.
DJ-1 is required for maintaining mitochondrial morphology and number of insulin secretory granules after MLDS treatment.
(a,b) Electron micrographs of islets from male control (a) and DJ-1 KO mice (b) after 4 weeks of MLDS treatment. (c,d) Image segmentation into mitochondria (red) and insulin secretory granules (green) using the trainable Weka Segmentation plugin for Fiji/ImageJ. Scale bar, 2 μm. (e,f) Quantification of total number of secretory granules (e) and mitochondrial area per section (f) in control and DJ-1 KO mice. n > 30 images per condition from n = 2 control and n = 3 DJ KO mice. Data are expressed as means ± SD.
Fig 5.
DJ-1 islet cell–autonomously protects from STZ-induced beta cell death in vitro.
(a-p) LSM live cell images of pancreatic islets from male control (a-d;i-l) and DJ-1 KO mice (e-h;m-p) stained with Hoechst (a,e,i and m), calcein AM (b,f, j and n), and ethidium homodimer-1 (c,g,k and o). Merged images are shown in d,h,l and p. Scale bar, 50 μm. (q) Quantification of dead cells of pancreatic islets from control and DJ-1 KO mice after 24 hours exposure to STZ treatment. Data are presented as ethidium homodimer-1 positive area per total cell nuclei area. For one experiment, islets harvested from 3–5 mice per genotype were pooled, treated and quantified. Statistical significance was assessed using the means of n = 3 independent experiments. *p<0.05 (Two-way ANOVA followed by Tukey’s multiple comparison test). Data are expressed as means ± S.D.
Fig 6.
DJ-1 islet cell-autonomously protects beta cells from cytokine-induced apoptosis.
(a-p) TUNEL labelling of pancreatic islets exposed to IL-1β, IFN-γ, and TNF-α for 24 hours. Islet sections from male control (a-d;i-l) and DJ-1 KO mice (e-h;m-p) showing staining for cell nuclei (DAPI, a,e,i and m), beta cells (insulin) and dead cells (TUNEL) (b,f,j and n), and alpha cells (glucagon) and dead cells (TUNEL) (c,g, k and o). Merged images are shown in d,h,l and p. Apoptotic cells are shown in red. Scale bar, 50 μm. (q) Quantification of TUNEL positive beta cells in pancreatic islets from control and DJ-1 KO mice after 24 hours exposure to IL-1β, IFN-γ, and TNF-α. The number of apoptotic beta cells per total number of beta cells is presented as percentage of untreated control. For one experiment, islets harvested from 3–5 mice per genotype were pooled, treated and quantified. Statistical significance was assessed using the means of n = 3 independent experiments. *p<0.05 (Two-way ANOVA followed by Tukey’s multiple comparison test). Data are expressed as means ± S.D.