mTOR signaling in liver regeneration: Rapamycin combined with growth factor treatment

Abstract

Aim: To investigate the effects of mammalian target of rapamycin (mTOR) inhibition on liver regeneration and autophagy in a surgical resection model.

Methods: C57BL/6 mice were subjected to a 70% partial hepatectomy (PH) and treated intraperitoneally every 24 h with a combination of the mTOR inhibitor rapamycin (2.5 mg/kg per day) and the steroid dexamethasone (2.0 mg/kg per day) in phosphate buffered saline (PBS) or with PBS alone as vehicle control. In the immunosuppressant group, part of the group was treated subcutaneously 4 h prior to and 24 h after PH with a combination of human recombinant interleukin 6 (IL-6; 500 μg/kg per day) and hepatocyte growth factor (HGF; 100 μg/kg per day) in PBS. Animals were sacrificed 2, 3 or 5 d after PH and liver tissue and blood were collected for further analysis. Immunohistochemical staining for 5-Bromo-2'-deoxyuridine (BrdU) was used to quantify hepatocyte proliferation. Western blotting was used to detect hepatic microtubule-associated protein 1 light chain 3 (LC3)-II protein expression as a marker for autophagy. Hepatic gene expression levels of proliferation-, inflammation- and angiogenesis-related genes were examined by real-time reverse transcription-polymerase chain reaction and serum bilirubin and transaminase levels were analyzed at the clinical chemical core facility of the Erasmus MC-University Medical Center.

Results: mTOR inhibition significantly suppressed regeneration, shown by decreased hepatocyte proliferation (2% vs 12% BrdU positive hepatocyte nuclei at day 2, P < 0.01; 0.8% vs 1.4% at day 5, P = 0.02) and liver weight reconstitution (63% vs 76% of initial total liver weight at day 3, P = 0.04), and furthermore increased serum transaminase levels (aspartate aminotransferase 641 U/L vs 185 U/L at day 2, P = 0.02). Expression of the autophagy marker LC3-II, which was reduced during normal liver regeneration, increased after mTOR inhibition (46% increase at day 2, P = 0.04). Hepatic gene expression showed an increased inflammation-related response [tumor necrosis factor (TNF)-α 3.2-fold upregulation at day 2, P = 0.03; IL-1Ra 6.0-fold upregulation at day 2 and 42.3-fold upregulation at day 5, P < 0.01] and a reduced expression of cell cycle progression and angiogenesis-related factors (HGF 40% reduction at day 2; vascular endothelial growth factor receptor 2 50% reduction at days 2 and 5; angiopoietin 1 60% reduction at day 2, all P ≤ 0.01). Treatment with the regeneration stimulating cytokine IL-6 and growth factor HGF could overcome the inhibitory effect on liver weight (75% of initial total liver weight at day 3, P = 0.02 vs immunosuppression alone and P = 0.90 vs controls) and partially reversed gene expression changes caused by rapamycin (TNF-α and IL-1Ra levels at day 2 were restored to control levels). However, no significant changes in hepatocyte proliferation, serum injury markers or autophagy were found.

Conclusion: mTOR inhibition severely impairs liver regeneration and increases autophagy after PH. These effects are partly reversed by stimulation of the IL-6 and HGF pathways.

Keywords: Autophagy; Hepatocyte proliferation; Microtubule-associated protein 1 light chain 3; Partial hepatectomy; Rapamycin.

Figure 1

Effects of mammalian target of rapamycin inhibition on body and liver weight. A: Harvest body weight at days 2, 3 and 5 after partial hepatectomy (PH) vs initial body weight; B: Harvest liver weight at days 2, 3 and 5 after PH vs total liver weight prior to PH. Data are shown as mean ± SE. BW: Body weight; R/D: Rapa-Dex; IL-6: Interleukin 6; HGF: Hepatocyte growth factor; PBS: Phosphate buffered saline.

Figure 2

Effects of mammalian target of rapamycin inhibition on hepatocyte proliferation. A, B: Livers were processed for immunohistochemistry on 5-Bromo-2’-deoxyuridine (BrdU) to quantify hepatocyte proliferation. A: Representative pictures (× 400) of hepatocyte proliferation at day 2 after partial hepatectomy (PH); B: Quantification of hepatocyte proliferation at day 2, 3 and 5 after PH; C, D: Hepatic gene expression levels of cyclin D1 and proliferating cell nuclear antigen (PCNA) were determined by quantitative reverse transcription-polymerase chain reaction and normalized against TATA binding protein. C: Expression levels of cyclin D1 at day 2 and 5 after PH; D: Expression levels of PCNA at day 2 and 5 after PH. Data are shown as mean ± SE. ^aP ≤ 0.05 vs phosphate buffered saline (PBS); ^cP ≤ 0.05 vs Rapa-Dex (R/D). HGF: Hepatocyte growth factor; IL-6: Interleukin 6.

Figure 3

Effects of partial hepatectomy and mammalian target of rapamycin inhibition on hepatic autophagy. Hepatic protein levels of the autophagy marker microtubule-associated protein 1 light chain 3 (LC3)-II were determined by Western blotting analysis and normalized against actin. A: Effects of liver resection on autophagy at day 2 after partial hepatectomy (PH); B: Western blotting showing effects of mammalian target of rapamycin inhibition on autophagy at day 2 after PH; C: Quantification of autophagy at day 2 and 5 after PH. Data are shown as mean ± SE. ^aP ≤ 0.05 vs phosphate buffered saline (PBS). R/D: Rapa-Dex; HGF: Hepatocyte growth factor; IL-6: Interleukin 6.

Figure 4

Effects of mammalian target of rapamycin inhibition on hepatocyte injury. Serum levels at day 2 after partial hepatectomy (PH) for aspartate aminotransferase (AST) (A), alanine aminotransferase (ALT) (B) and bilirubin (C); D: Histologic changes (× 400) at day 5 after PH in liver tissue from Rapa-Dex (R/D) treated animals. Data are shown as mean ± SE. ^aP ≤ 0.05 vs phosphate buffered saline (PBS). HGF: Hepatocyte growth factor; IL-6: Interleukin 6.

Figure 5

Effects of mammalian target of rapamycin inhibition on inflammation and cell cycle related gene expression. Hepatic gene expression levels were determined by quantitative reverse transcription-polymerase chain reaction and normalized against TATA binding protein. A: Expression levels of tumor necrosis factor α (TNF-α) at day 2 and 5 after partial hepatectomy (PH); B: Expression levels of interleukin 1 receptor antagonist (IL-1Ra) at day 2 and 5 after PH; C: Expression levels of interleukin 6 (IL-6) at day 2 and 5 after PH; D: Expression levels of hepatocyte growth factor (HGF) at day 2 and 5 after PH; E: Expression levels of transforming growth factor β (TGF-β) at day 2 and 5 after PH. Data are shown as mean ± SE. ^aP ≤ 0.05 vs phosphate buffered saline (PBS); ^cP ≤ 0.05 vs Rapa-Dex (R/D).

Figure 6

Effects of mammalian target of rapamycin inhibition on angiogenic gene expression. Hepatic gene expression levels were determined by quantitative reverse transcription-polymerase chain reaction and normalized against TATA binding protein. A: Expression levels of vascular endothelial growth factor receptor 2 (VEGF-R2) at day 2 and 5 after partial hepatectomy (PH); B: Expression levels of angiopoietin 1 (Ang-1) at day 2 and 5 after PH; C: Expression levels of VEGF-A at day 2 and 5 after PH; D: Expression levels of VEGF-R1 at day 2 and 5 after PH. Data are shown as mean ± SE. ^aP ≤ 0.05 vs phosphate buffered saline (PBS); ^cP ≤ 0.05 vs Rapa-Dex (R/D). HGF: Hepatocyte growth factor; IL-6: Interleukin 6.