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HEPATOCYTE-SPECIFIC DELETIONS OF AUTOPHAGY-INITIATING GENES RESULT IN PRONOUNCED HEPATOMEGALY AND PATHOLOGY

Date
May 20, 2024

Background: Canonical autophagy is a multi-step process and involves initiation (mediated by Fip200, Ulk1, etc.), phagophore nucleation (Atg14, Becn1, etc.), followed by autophagosome formation (Atg16l1, Atg5, Atg7, etc.), and completes with autolysosome digestion. Autophagy protects the liver against injury and disease. Previous studies showed that hepatocyte conditional knockout (cKO) of Atg5 or Atg7 genes in autophagosome formation, resulted in hepatomegaly. However, it is unclear whether genes in the upstream autophagy process such as Fip200 or Atg14 are involved in modulating liver size and pathology.
Methods: We generated Fip200, Atg14, Atg16l1, and Atg7 hepatocyte cKO mice. Fip200 and Atg14 cKO mice were additionally crossed with p62 or Yap KO mice. Hepatocyte size, cell death, proliferation, and Kupffer cell infiltration were defined by using beta-catenin, cleaved caspase-3, Ki67, and F4/80 immunostaining, respectively. Cell death inhibition was performed by administering inhibitors for apoptosis (Z-VAD-FMK), necroptosis (necrostatin-1), and pyroptosis (Ac-FCTD-CMK). Anti-CSF1R and anti-Ly6G antibodies were administrated to deplete macrophages and neutrophils.
Results: Fip200 and Atg14 cKO mice showed more accelerated and profound hepatomegaly compared to the Atg16l1 and Atg7 cKO mice. Hepatomegaly was not due to developmental defects, as the phenotype was induced after these genes were deleted post-weaning. Hepatic Fip200 and Atg14 cKO mice increased hepatocyte size, hepatocyte proliferation and death, and Kupffer cell infiltration. In contrast, livers from the Atg16l1 and Atg7 cKO mice showed relatively unremarkable histology. Liver histologic changes in Atg14 cKO mice were not rescued by either cell death inhibition or depletion of macrophages and neutrophils. Furthermore, in contrast to previous studies where concomitant deletion of either Yap or p62 was able to reverse hepatomegaly and liver pathology in Atg7 or Atg5 cKO mice, in the Fip200 and Atg14 cKO mice, deletion of Yap did not rescue liver size or pathology, while p62 deletion rescued liver size but only partially rescued histology changes.
Conclusion: Compared to Atg16l1 and Atg7 cKO, Fip200 and Atg14 cKO result in aggravated hepatomegaly, accompanied by increases in hepatocyte proliferation and death, and Kupffer cell infiltration. Phenotypes associated with the Fip200 and Atg14 cKO mice are only partially rescued by concomitant p62 KO but not by Yap KO. Our results suggest that genes involved in autophagy initiation have more profound effects on liver pathophysiology compared to genes associated with autophagosome formation.