Subnormothermic Perfusion in the Isolated Rat Liver Preserves the Antioxidant Glutathione and Enhances the Function of the Ubiquitin Proteasome System

Abstract

The reduction of oxidative stress is suggested to be one of the main mechanisms to explain the benefits of subnormothermic perfusion against ischemic liver damage. In this study we investigated the early cellular mechanisms induced in isolated rat livers after 15 min perfusion at temperatures ranging from normothermia (37°C) to subnormothermia (26°C and 22°C). Subnormothermic perfusion was found to maintain hepatic viability. Perfusion at 22°C raised reduced glutathione levels and the activity of glutathione reductase; however, lipid and protein oxidation still occurred as determined by malondialdehyde, 4-hydroxynonenal-protein adducts, and advanced oxidation protein products. In livers perfused at 22°C the lysosomal and ubiquitin proteasome system (UPS) were both activated. The 26S chymotrypsin-like (β5) proteasome activity was significantly increased in the 26°C (46%) and 22°C (42%) groups. The increased proteasome activity may be due to increased Rpt6 Ser120 phosphorylation, which is known to enhance 26S proteasome activity. Together, our results indicate that the early events produced by subnormothermic perfusion in the liver can induce oxidative stress concomitantly with antioxidant glutathione preservation and enhanced function of the lysosomal and UPS systems. Thus, a brief hypothermia could trigger antioxidant mechanisms and may be functioning as a preconditioning stimulus.

Figure 1

Temperature effects on the hepatic function of isolated perfused rat (IPR) livers. IPR livers were perfused in a nonrecirculating system with Krebs-Henseleit buffer at 37°C for 15 min, for stabilization, and then at the indicated temperatures for 15 min more. Results show cell viability by ALT assay in the perfusate (a) and NO production in liver homogenates (b). Values expressed as mean ± SEM of n = 6. Significant differences from livers perfused at 37°C: ^∗ P < 0.05 and ^∗∗ P < 0.01.

Figure 2

Lipid and protein oxidation in isolated perfused rat livers. IPR livers were perfused in a nonrecirculating system with Krebs-Henseleit buffer at 37°C for 15 min, for stabilisation, and then at the indicated temperatures for 15 min more. Lipid peroxidation was measured as TBARS (a) and by western blotting of HNE-protein adducts (b), and advanced oxidation protein products (dityrosine-containing and cross-linking protein products) measured spectrophotometrically at 340 nm (c). Values expressed as mean ± SEM of n = 6. Significant differences from livers perfused at 37°C: ^∗ P < 0.05. Significant differences from livers perfused at 26°C: ⁺⁺ P < 0.01.

Figure 3

Temperature effects on antioxidant status in isolated perfused rat livers. IPR livers were perfused at 37°C for 15 min and then at the indicated temperatures for 15 min more. Bar graphs show hepatic GSH (a), GSH/GSSG ratio (b), and GSH-reductase activity (c). Values expressed as mean ± SEM of n = 6. Significant differences from livers perfused at 37°C: ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001. Significant differences from livers perfused at 26°C: ⁺⁺ P < 0.01 and ⁺⁺⁺ P < 0.001.

Figure 4

Lysosomal activity in isolated perfused rat livers. IPR livers were perfused at 37°C for 15 min and then at the indicated temperatures for 15 min more. To independent lysosomal enzymes, cathepsin B (a) and cathepsin L (b) were measured to determine if lysosomal activity was affected. Data is expressed as mean ± SEM of n = 6. Significant differences from livers perfused at 37°C: ^∗ P < 0.05. Significant differences from livers perfused at 26°C: ⁺ P < 0.05.

Figure 5

Proteasomal activity of 26S proteasome in isolated perfused rat livers. IPR livers were perfused at 37°C for 15 min and then at the indicated temperatures for 15 min more. The caspase-like β1 (a), trypsin-like β2 (b), and chymotrypsin-like β5 (c) activities of the proteasome were determined. Data is expressed as mean ± SEM of n = 6. Significant differences from livers perfused at 37°C: ^∗∗ P < 0.01.

Figure 6

Expression of ubiquitin proteasome system in isolated perfused rat livers. IPR livers were perfused at 37°C for 15 min and then at the indicated temperatures for 15 min more. Results show western blots and densitometric analysis for Rpt1 19S (a) and 20S core subunit (b). (c) Levels of polyubiquitinated proteins; densitometry includes all polyubiquitinated bands detected. β-Actin was used as a normalization control for the western blots. (d) Ponceau S (total protein) staining was used as loading control for phospho S120 Rpt6 and Rpt6 densitometric analyses of western blots; values were then used to calculate p-Rpt6/Rpt6 ratios. Results are expressed as mean ± SEM of n = 3 to 6 independent samples per group. Significant differences from livers perfused at 37°C: ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001.