Reduced peroxisomal function increases insulin secretion, promotes insulin oxidation, and impairs β cell maturity

Figures

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  Figure 1. Pancreas- and β cell–specific deletion of Pex5 impairs peroxisomal matrix protein import and function. (A) Pex5 mRNA abundance by RT-PCR from Pex5CON and Pex5Pdx1–/– mice; n = 7–14. (B) Pex5 abundance by immunoblot in pancreatic lysates and (C) isolated islets; n = 3–4. (D) Pancreatic tissue from Pex5CON (top panels) and Pex5Pdx1–/– (bottom panels) mice costained for PMP70 (cyan), insulin (green), and Hoechst (gray); n = 3 with representative image shown. Scale bars:100 μm (columns 1–3) and 20 μm (last column is a higher-resolution image of tissue denoted by yellow box in 3rd column). (E) Pancreatic tissue costained for Acox1 (green), PMP70 (red), insulin (blue), and Hoechst (gray); n = 3 with representative image shown, scale bar: 20 μm (columns 1–3) and 10 μm (last column is a higher-resolution image of tissue denoted by yellow box). (F) Volcano plot for ether lipids in pancreatic tissue from 30-week-old mice represented as Pex5Pdx1–/–/Pex5CON; n = 9–11. (G) Serum triglyceride content in 15-week-old mice; n = 8–10. (H) ITT in 10–16-week-old mice; n = 8. (I) Pex5 mRNA abundance by RT-PCR from Pex5CON and Pex5Ins1–/– mice; n = 6–8. (J) Pex5 abundance by immunoblot in isolated…

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  Figure 2. Peroxisomes are critical for maintenance of glucose tolerance and β cell function in male mice. (A) Fed blood glucose values in male Pex5CON, Pex5Pdx1–/–, and Pex5Ins1–/– mice from 8 to 30 weeks of age; n = 26–34. (B–E) i.p. GTTs in 8- (n = 9–14), 12- (n = 22), 20- (n = 15–16), and 30- (n = 8–19) week-old mice. (F) Oral GTT in 15-week-old mice; n = 11–25. (G–I) Fasting blood glucose, glucose infusion rate, and plasma insulin during a hyperglycemic clamp in 18-week-old mice; n = 4. (J–M) Glucose-mediated (16.7 mM glucose; G 16.7) and KCl-mediated (20 mM; KCl 20) insulin secretion and corresponding AUC calculations in isolated islets from 12- and 30-week-old mice, respectively; n = 6. (N) Insulin content in islets used for experiments in J–M; n = 6–13. (O) i.p. GTT in 30-week-old male Pex5CON and Pex5Ins1–/– mice; n = 29–31. (P) Oral GTT in 42-week-old mice; n = 9. (Q and R) Glucose-mediated (16.7 mM glucose; G 16.7) and KCl-mediated (20 mM; KCl 20) insulin secretion and corresponding AUC calculation in isolated islets from 30-week-old mice; n = 4. (S) Insulin content in islets used for the experiment in Q and R. (T) GSIS (2 mM versus 20 mM glucose) in 832/13 cells treated for 48…

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  Figure 3. Reduced peroxisomal function in the β cells of female mice has no impact on β cell function. (A) Fed blood glucose values in female Pex5CON, Pex5Pdx1–/–, and Pex5Ins1–/– mice from 12 to 36 weeks of age; n = 12–23. (B–D) Intraperitoneal GTTs in 12-week-old (n = 10–20), 20-week-old (n = 10–20), and 34-week-old (n = 10–20) mice, respectively. (E) Oral GTT in 36-week-old mice; n = 8–11. (F and G) Glucose- (16.7 mM glucose; G 16.7) and KCl- (20 mM; KCl 20) mediated insulin secretion, and corresponding AUC calculation, in isolated islets from 36-week-old mice; n = 8. (H) Insulin content in islets used for experiment in F and G. ns, not significant; *P < 0.05. P values shown on graph represent AUC for Pex5CON versus Pex5Pdx1–/– groups. Multiple unpaired t tests (A), 1-way ANOVA with multiple comparisons (G), or 2-tailed Student’s t test (H); wo, weeks old.

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  Figure 4. Reduced peroxisomal function in the pancreas of male mice has no impact on islet architecture or hormone content. (A) Pancreatic tissue from 30-week-old male Pex5CON and Pex5Pdx1–/– mice costained for insulin (green), glucagon (red), and Hoechst (blue), n = 5 with representative image shown, scale bar: 100 μm. (B and C) Pancreas mass (n = 5–10) and β cell mass (n = 5), respectively, in 30-week-old mice. (D and E) Insulin-positive area and islet fraction, respectively, in 30 week-old mice; n = 6–14. (F) Fasting serum insulin in 30-week-old mice; n = 15. (G) Fasting serum glucagon in 30-week-old mice; n = 7–22. One-way ANOVA (B–G).

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  Figure 5. Loss of peroxisomal function correlates with increased oxidative phosphorylation pathway activity, palmitate oxidation, and ATP content in male mice. (A–C) Volcano plot, KEGG pathway analysis, and upregulated genes in the oxidative phosphorylation pathway, respectively, determined from RNA-Seq data in islets from 22-week-old male Pex5CON and Pex5Pdx1–/– mice; n = 3–4. (D) Mitochondrial versus nuclear DNA content in isolated islets from 22-week-old mice; n = 7–10. (E) CPT1 activity in pancreatic mitochondrial isolates from 30-week-old mice; n = 7–8. (F and G) Palmitate oxidation to CO2, or acid-soluble metabolites (ASM), respectively, in the basal state, in the presence of etomoxir (ETX) or pyruvate (PYR) in pancreatic homogenates from 30-week-old mice; n = 12–13. (H and I) Pyruvate and leucine oxidation, respectively, in pancreatic homogenates from 30-week-old mice; n = 13–14. (J) Intracellular ATP content in isolated islets from 30-week-old mice treated with glucose (2, 11, or 20 mM); n = 4–9. (K) Intracellular ATP in 832/13 cells treated for 48 hours with siCTRL or siPex5 at 11 mM glucose; n = 4 individual experiments. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.…

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  Figure 6. Reduction in CPT1a abundance in peroxisomal-deficient male mice restores β cell GSIS and islet ATP content to control levels. (A) mRNA abundance by RT-PCR in islets from male Pex5/Cpt1aCON and Pex5/Cpt1aPdx1–/– mice; n = 7–8. (B) Pex5 abundance by immunoblot in isolated islets (top) and CPT1a abundance in pancreatic tissue (bottom), n = 4, scale bar: 50 μm. (C) mRNA abundance by RT-PCR in liver and scWAT subcutaneous White Adipose Tissue (scWAT); n = 10. (D) ITT in 12-week-old mice; n = 14–18. (E) GTT (i.p.) in 14-week-old mice; n = 24–36. (F) GTT (i.p.) in 26-week-old mice; n = 8–16. (G) Serum insulin from blood taken at 0-, 15-, and 60-minute time points during GTT shown in F; n = 12–16. (H–J) Fasting blood glucose, glucose infusion rate, and plasma insulin, respectively, during hyperglycemic clamp in 18-week-old mice; n = 4–5. (K and L) Glucose-mediated (16.7 mM glucose; G 16.7) and KCl-mediated (20 mM; KCl 20) insulin secretion and corresponding AUC calculation in islets from 15-week-old mice; n = 4. (M) Insulin content in islets used for the experiment in K and L. (N) mRNA abundance by RT-PCR in islets; n = 7–8. (O–R) Insulin-positive area, islet fraction, β cell mass, and…

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  Figure 7. Loss of functional peroxisomes correlates with increased abundance of posttranslationally modified insulin 1 and 2 and a truncated insulin 2 peptide in male Pex5Pdx1–/– mice. (A) Liquid chromatography (LC) chromatogram of insulin 1 eluting at 15.4–16 minutes and insulin 2 eluting at 16–17 minutes. (B) Entire mass spectrum revealing all insulin 1 and 2 peaks at different charge states and the arrival time distribution of peaks at 966.4 m/z and 1,159.5 m/z. (C–E) Mass spectra and arrival time distributions of insulin 1 and oxidized insulin 1; insulin 2 and oxidized insulin 2; and insulin 2: A+B(1–25) fragment, respectively. (F) Ion abundance of oxidized insulin 1, (G) percentage ion abundance of oxidized insulin 1/insulin 1, (H) ion abundance of oxidized insulin 2, (I) percentage ion abundance of oxidized insulin 2/insulin 2, (J) ion abundance of insulin 2: A+B(1–25) fragment, and (K) percentage ion abundance of insulin 2: A+B(1–25) fragment/insulin 2, in islets from 30-week-old Pex5CON and Pex5Pdx1–/– mice. n = 4 individual islets per genotype. *P < 0.05. 2-tailed Student’s t test (F–K).

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  Figure 8. Coordinated alterations in oxidative stress signature in pancreas and islets from Pex5Pdx1–/– mice. (A–C) Volcano plots for identified metabolites including (D) 8-hydroxy-2′-deoxyguanosine, and (E) 4-hydroxynonenal in pancreatic tissue from 12-week-old male (A), 30-week-old male (B), and 36-week-old female (C) Pex5CON and Pex5Pdx1–/– mice; n = 9–11. (F) Representative EPR spectra of carbon-centered lipid-derived radical adducts (POBN-LOO•) in pancreatic tissue from 20-week-old male mice and (G) bar graphs show EPR signal intensities (arbitrary units) of the measured peak amplitudes normalized to 300 mg tissue. (H) Protein carbonylation in islets from 28-week-old male mice. (I and J) Gene expression, expressed as normalized counts, for the (I) Sod1 and (J) Gpx1 genes in islets from 22-week-old male mice. (F–J) n = 4–6 per genotype. *P < 0.05, **P < 0.01. One-way ANOVA with multiple comparisons (D and E), or 2-tailed Student’s t test (G–J); wo, weeks old.

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  Figure 9. Global shifts in transcriptional signature in islets from male Pex5Pdx1–/– mice. (A) Change in expression of β cell and exocrine markers determined from RNA-Seq data in islets from 22-week-old Pex5CON and Pex5Pdx1–/– mice; n = 3–4. (B) Pancreatic tissue from 15-week-old (left panel) and 30-week-old (right panel) mice costained for insulin (green), Nkx6.1 (red), and Hoechst (blue). (C) Pancreatic tissue from 6–8-week-old, 15-week-old, and 30-week-old mice costained for insulin (green), ALDH1A3 (red), and Hoechst (blue). (D) Pancreatic tissue from 15-week-old, 30-week-old, and 52-week-old mice costained for insulin (green), ALDH1A3 (red), and Hoechst (blue). (B–D) n = 5 with representative image shown, scale bars: 100 μm; wo, weeks old.

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  Figure 10. Loss of functional peroxisomes in pancreatic tissue promotes β cell dedifferentiation in female mice. (A) RNA-Seq data in islets from 36-week-old female Pex5CON and Pex5Pdx1–/– mice; n = 4. (B) Npy and Nptx2 mRNA abundance by RT-PCR in islets; n = 7. (C) Pancreatic tissue from 36-week-old mice costained for insulin (green), ALDH1A3 (red), and Hoechst (blue), n = 5 with representative image shown, scale bar: 100 μm. (D and E) KEGG pathway analysis and expression of genes in the glycerolipid metabolism pathway, respectively, determined from RNA-Seq data in islets from 36-week-old mice; n = 4. (F–H) Pnliprp1, Reg1, and Reg3a mRNA abundance, respectively, by RT-PCR in islets from male (n = 9) and female (n = 7) mice. *P < 0.05, ***P < 0.001, ****P < 0.0001. One-way ANOVA with multiple comparisons (B and F–H).