O2166: Autophagy impairment mediated by mTORC1 activation in hepatocytes correlates with liver injury in a preclinical model of short bowel syndrome

Background: Autophagy is the destruction of damaged or redundant cellular components and is critical for both cellular preservation and maintenance. Dysregulated autophagy is involved in the development of various liver injury models. However, little is known about this process in intestinal failure-associated liver disease.

Methods: C57BL/6 male and female mice were randomized to either 50-75% proximal small bowel resection (SBR) or sham operations and sacrificed 2 or 10 weeks postoperatively. At least six mice were in each cohort’s operative stratification (see Figure 1A for exact cohort numbers). Mice were fed a high-fat liquid diet. Liver injury was assessed by serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations. Autophagy was first assessed by analyzing p62 expression via qRT-PCR, Western blots, and immunohistochemistry (IHC). p62 is inversely related to autophagy function. Primary hepatocytes from a subset of 2-week male mice (2 shams and 2 SBR) were isolated and autophagy was further verified via co-staining with Tetramethylrhodamine, methyl ester (TMRM) and GFP-LC3. Mammalian target of rapamycin complex 1 (mTORC1) activity was measured via p-S6 western blot and IHC on liver tissue. PCR and serum results were compared with an unpaired t-test using Prism software. Studies were approved by the institution’s Animal Studies Committee (Protocol #20-0197).

Results: SBR mice demonstrate significant liver injury compared to shams indicated by elevated serum AST and ALT levels (Figure 1A), all p< 0.05. Autophagy impairment in SBR liver is revealed by increased transcription of p62 for each time/sex cohort, all p< 0.05 (Figure 1B). This finding is confirmed by Western Blot and IHC studies. Representative images demonstrate increased translation of p62 at both 2 and 10 weeks in SBR mice (Figure 2 C, D). Autophagy impairment is further verified in primary hepatocytes isolated from 2-week mice. When autophagy is stimulated via starvation, sham mice show evidence of lipophagy, characterized by the GFP-LC3 (green) staining surrounding lipid droplets (Figure 1E). In contrast, the GFP-LC3 staining in SBR hepatocytes is localized around the nucleus, suggesting upstream inhibition of autophagosome vesicle formation – a prerequisite to autophagy. Additionally, because uptake of TMRM (red) staining is dependent on the mitochondrial membrane potential, the relatively low intensity noted in SBR hepatocytes is suggestive of mitochondrial dysfunction (Figure 1E). Finally, an important upstream regulator of autophagy is the mTORC1 pathway. At each time point, SBR livers exhibit evidence of increased mTORC1 activity assessed by enhanced pS6 protein expression via western blots and IHC (Figure 2 A,B).

Conclusion: Autophagy is dysregulated in the hepatocytes of injured SBR livers. This impairment is persistent at both 2 and 10 weeks. Autophagy dysregulation is correlated with mTOR activation in SBR mice. Interventions to restore or activate autophagy may ameliorate this specific liver disease. Future work includes pharmaceutically inhibiting the mTOR pathway.