Regulation of Liver Regeneration by hepatocyte O-GlcNAcylation in mice

Abstract

The liver has a unique capacity to regenerate after injury in a highly orchestrated and regulated manner. Here we report that O-GlcNAcylation, an intracellular posttranslational modification (PTM) regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a critical termination signal for liver regeneration (LR) following partial hepatectomy (PHX). We studied liver regeneration after PHX on hepatocyte specific OGT and OGA knockout mice (OGT-KO and OGA-KO), which caused a significant decrease (OGT-KO) and increase (OGA-KO) in hepatic O-GlcNAcylation, respectively. OGA-KO mice had normal regeneration, but the OGT-KO mice exhibited substantial defects in termination of liver regeneration with increased liver injury, sustained cell proliferation resulting in significant hepatomegaly, hepatic dysplasia and appearance of small nodules at 28 days after PHX. This was accompanied by a sustained increase in expression of cyclins along with significant induction in pro-inflammatory and pro-fibrotic gene expression in the OGT-KO livers. RNA-Seq studies revealed inactivation of hepatocyte nuclear 4 alpha (HNF4α), the master regulator of hepatic differentiation and a known termination signal, in OGT-KO mice at 28 days after PHX, which was confirmed by both Western blot and IHC analysis. Furthermore, a significant decrease in HNFα target genes was observed in OGT-KO mice, indicating a lack of hepatocyte differentiation following decreased hepatic O-GlcNAcylation. Immunoprecipitation experiments revealed HNF4α is O-GlcNAcylated in normal differentiated hepatocytes. These studies show that O-GlcNAcylation plays a critical role in the termination of LR via regulation of HNF4α in hepatocytes.

The Essential Role of O-GlcNAcylation in Hepatic Differentiation

“…Additionally, O-GlcNAcylation is found to regulate serum response factor (SRF), which leads to the activation of hepatic stellate cells and fibrosis (42). Additionally, our data corroborate other studies showing that O-GlcNAcylation is a critical regulator of cell proliferation (2,(43)(44)(45). Further, our data exhibited increased stemness gene expression, indicating a more stem cell-like identity.…”

“…Mice were euthanized two months after AAV8 injection. In all studies, liver injury was assessed by serum ALT activity assay, and livers were weighed after cholecystectomy to calculate liver-weight-to-body-weight ratios as previously described (2). Glucose was measured in serum according to the manufacturer's protocol (Pointe Scientific cat# G7521120).…”

“…O-GlcNAcylation has been shown to be a critical regulator in cellular differentiation, such as hematopoietic stem cells and neuronal cells (9,10). Previous studies from our lab have shown cross-talk between O-GlcNAcylation and HNF4α (2). Here, we studied the effects of chronic loss of O-GlcNAcylation and the role of O-GlcNAcylation in the progression of liver disease.…”

“…Because the hexosamine biosynthetic pathway, which produces UDP-GlcNAc, integrates multiple metabolic pathways, including nucleotide, fatty acid, protein, and glucose metabolism (1), changes in multiple independent metabolic pathways can affect O-GlcNAcylation. Previous studies show that O-GlcNAcylation plays a role in a number of cellular processes, including metabolism, inflammation, and cell proliferation (1,2). Abnormal O-GlcNAcylation levels have been linked to various diseases, including cancer (1,3,4).…”

“…Abnormal O-GlcNAcylation levels have been linked to various diseases, including cancer (1,3,4). A recent study by our group found that a lack of hepatic O-GlcNAcylation during liver regeneration impairs the termination phase and leads to sustained cell proliferation and loss of hepatocyte identity (2). Whereas O-GlcNAcylation is known to be involved in hepatic fibrosis and HCC pathogenesis, the mechanisms are not clear (4)(5)(6).…”