Ceruloplasmin and hephaestin jointly protect the exocrine pancreas against oxidative damage by facilitating iron efflux

Abstract

Little is known about the iron efflux from the pancreas, but it is likely that multicopper ferroxidases (MCFs) are involved in this process. We thus used hephaestin (Heph) and ceruloplasmin (Cp) single-knockout mice and Heph/Cp double-knockout mice to investigate the roles of MCFs in pancreatic iron homeostasis. We found that both HEPH and CP were expressed in the mouse pancreas, and that ablation of either MCF had limited effect on the pancreatic iron levels. However, ablation of both MCFs together led to extensive pancreatic iron deposition and severe oxidative damage. Perls' Prussian blue staining revealed that this iron deposition was predominantly in the exocrine pancreas, while the islets were spared. Consistent with these results, plasma lipase and trypsin were elevated in Heph/Cp knockout mice, indicating damage to the exocrine pancreas, while insulin secretion was not affected. These data indicate that HEPH and CP play mutually compensatory roles in facilitating iron efflux from the exocrine pancreas, and show that MCFs are able to protect the pancreas against iron-induced oxidative damage.

Keywords: Ceruloplasmin; Hephaestin; Iron efflux; Multicopper ferroxidase; Oxidative damage; Pancreas.

Graphical abstract

Fig. 1

CP and HEPH protein and mRNA expression in the pancreas. (A) Expression of CP and HEPH protein in lysates from the pancreas of WT, Heph/Cp KO, Cp KO, and Heph KO mice (n = 4 per group). Tubulin was used as a loading control. The western blot signals were quantified, and values were normalized to tubulin expression and expressed as a proportion of the WT control. (B) Quantitative real-time PCR analysis of Cp and Heph mRNA expression in the pancreas of WT, Heph/Cp KO, Cp KO, and Heph KO mice (n = 6 per group). Values were normalized to GAPDH expression and expressed as a proportion of the WT control value. Data are shown as mean ± SEM. Bars without common letters are significantly different (p < 0.05, as determined by one way ANOVA followed by Tukey's multiple comparisons test).

Fig. 2

Heph/Cp KO mice exhibit severe iron overload in the pancreas. (A) Expression of ferritin protein in lysates from the pancreas of WT, Heph/Cp KO, Cp KO, and Heph KO mice (n = 4 per group). Tubulin was used as a loading control. The western blot signals were quantified, and values were normalized to tubulin expression and expressed as a proportion of the WT control value. (B) Non-heme iron concentration of the pancreas of WT, Heph/Cp KO, Cp KO, and Heph KO mice (n = 6 per group). (C) Macroscopic appearance of the pancreas of WT, Heph/Cp KO, Cp KO, and Heph KO mice. Data are shown as mean ± SEM. Bars without common letters are significantly different (p < 0.05, as determined by one way ANOVA followed by Tukey's multiple comparisons test) (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.).

Fig. 3

Expression of the mRNAs encoding iron-related proteins and proinflammatory cytokines in the pancreas. Fpn1(+IRE), Hamp, Dmt1(+IRE), Tfrc, IL-1β, IL-6, and TNF-α mRNA expression in the pancreas of WT, Heph/Cp KO, Cp KO, and Heph KO mice is shown (n = 6 per group). Values were normalized to GAPDH expression and expressed as a proportion of the WT control value. Data are shown as mean ± SEM. Bars without common letters are significantly different (p < 0.05, as determined by one way ANOVA followed by Tukey's multiple comparisons test).

Fig. 4

Iron deposition predominates in the exocrine pancreas of Heph/Cp KO mice. (A and B) Representative images of Perls’ Prussian blue staining for iron in the exocrine (A) and endocrine (B) pancreas of WT, Heph/Cp KO, Cp KO, and Heph KO mice (n = 4 per group). In each part of the figure, the upper row is of lower magnification than the lower row.

Fig. 5

Heph/Cp KO mice exhibit manifestations of exocrine pancreatic injury and inflammatory infiltration. (A and B) Representative images of TUNEL staining for apoptotic cells (A) and CD68 staining for macrophages (B) in the exocrine pancreas of WT, Heph/Cp KO, Cp KO, and Heph KO mice (n = 4 per group). (C) Plasma lipase and trypsin activities of WT, Heph/Cp KO, Cp KO, and Heph KO mice (n = 6 per group). Data are shown as mean ± SEM. Bars without common letters are significantly different (p < 0.05, as determined by one way ANOVA followed by Tukey's multiple comparisons test).

Fig. 6

Insulin levels are not altered in Heph/Cp KO mice. (A) Representative images of insulin staining in islets of WT, Heph/Cp KO, Cp KO, and Heph KO mice (n = 4 per group). (B) Transmission electron micrographs of insulin granules in β-cells of WT, Heph/Cp KO, Cp KO, and Heph KO mice (n = 4 per group).