IGF-1R contributes to stress-induced hepatocellular damage in experimental cholestasis

Abstract

The insulin-like growth factor type 1 receptor (IGF-1R) controls aging and cellular stress, both of which play major roles in liver disease. Stimulation of insulin-like growth factor signaling can generate cell death in vitro. Here, we tested whether IGF-1R contributes to stress insult in the liver. Cholestatic liver injury was induced by bile duct ligation in control and liver-specific IGF-1R knockout (LIGFREKO) mice. LIGFREKO mice displayed less bile duct ligation-induced hepatocyte damage than controls, while no differences in bile acid serum levels or better adaptation to cholestasis by efflux transporters were found. We therefore tested whether stress pathways contributed to this phenomenon; oxidative stress, ascertained by both malondialdehyde content and heme oxygenase-1 expression, was similar in knockout and control animals. However, together with a lower level of eukaryotic initiation factor-2 alpha phosphorylation, the endoplasmic reticulum stress protein CHOP and its downstream pro-apoptotic target Bax were induced to lesser extents in LIGFREKO mice than in controls. Expression levels of cytokeratin 19, transforming growth factor-beta1, alpha-smooth muscle actin, and collagen alpha1(I) in LIGFREKO mice were all lower than in controls, indicating reduced ductular and fibrogenic responses and increased cholestasis tolerance in these mutants. This stress resistance phenotype was also evidenced by longer post-bile duct ligation survival in mutants than controls. These results indicate that IGF-1R contributes to cholestatic liver injury, and suggests the involvement of both CHOP and Bax in this process.

Figure 1

Changes of hepatic Igf1r, Igf1, and Igf2 expression associated with cholestatic liver injury. Liver samples from Sham or BDL control mice were examined 3 or 21 days after surgery by RT-QPCR for expression of Igf1r (A), Igf1 (B), and Igf2 (C). Data were normalized to 18S rRNA and represent means ± SEM for 4 to 6 animals. *P < 0.05. NS, not significant.

Figure 2

Cholestasis and liver injury in LIGFREKO mice. Blood samples were collected from Sham or BDL, control and LIGFREKO mice on postoperative day 3. Panels show serum concentrations of (A) bile acids, (B) total bilirubin, (C) aspartate aminotransferase (ASAT), and (D) alanine aminotransferase (ALAT). Data represent means ± SEM for 5 to 10 animals. *P < 0.05; **P < 0.01; ***P < 0.005.

Figure 3

Bile infarcts in LIGFREKO mice. Liver samples were collected from Sham or BDL, control and LIGFREKO mice on postoperative day 3, stained for H&E and for TUNEL. A: Liver was morphologically normal in control and LIGFREKO sham groups. After BDL, bile infarcts (arrows) were smaller in LIGFREKO than in control mice. Clusters of TUNEL-positive hepatocytes were observed only in bile infarct areas (asterisk). Micrographs are representative of 5 to 10 animals in each group. B: Infarct area was assessed by digital image analysis. Data are expressed as a percentage of whole sections and represent means ± SEM for 10 animals. *P < 0.05.

Figure 4

Post-BDL changes in efflux ABC transporters in LIGFREKO. Liver samples were collected from Sham or BDL, control and LIGFREKO mice on postoperative day 3 and were examined for (A) Mrp3, (B) Mrp4, and (C) bile salt export pump expression by RT-QPCR. Data were normalized to 18S rRNA and represent means ± SEM for 5 to 6 animals. NS, not significant; *P < 0.05; ***P < 0.005.

Figure 5

Induction and IGF-1R regulation of major stress pathways associated with cholestatic liver injury. Liver samples were collected from Sham or BDL, control and LIGFREKO mice on postoperative day 3 and were examined. A: JNK activation determined by western blotting for p-JNK and total JNK protein. Blots are representative of groups of 5 animals. B: GRP78 expression measured by RT-QPCR. C: CHOP expression determined by Western blot (upper, representative blot) and RT-QPCR (lower). D: Bax expression measured by RT-QPCR. Data were normalized to 18S rRNA and represent means ± SEM for 5 to 8 animals. *P < 0.05; **P < 0.01; ***P < 0.005. E: eIF2α activation determined by Western blot analysis of p-eIF2α and total eIF2α proteins. Blots are representative of groups of 5 animals.

Figure 6

Ductular and fibrogenic response to cholestatic liver injury in LIGFREKO mice. Liver samples were collected from Sham or BDL, control and LIGFREKO mice on postoperative day 3 and were examined by RT-QPCR for expression of (A) CK19, (B) TGF-β1, (C) α-SMA, and (D) Col1A1. We performed the same experiments as in (A) to (D) for animals at 21 days post-BDL (E–H). Data were normalized to 18S rRNA and represent means ± SEM for 6 to 8 animals. *P < 0.05; **P < 0.01; ***P < 0.005.

Figure 7

Impact of IGF-1R on survival in BDL mice. Survival after BDL was monitored for 21 days and censored thereafter. Kaplan-Meier survival curves for 9 control and 8 LIGFREKO mice were compared (P < 0.05, log-rank test).