Dataset Information

O-GlcNAcylation Coordinates Glutaminolysis by Regulating the Stability and Membrane Trafficking of ASCT2 in Hepatic Stellate Cells.

ABSTRACT:

Background and aims

Recognition of excessive activation of hepatic stellate cells (HSCs) in liver fibrosis prompted us to investigate the regulatory mechanisms of HSCs. We aimed to examine the role of O-GlcNAcylation modification of alanine, serine, cysteine transporter 2 (ASCT2) in HSCs and liver fibrosis.

Methods

The expression of O-GlcNAcylation modification in fibrotic mice livers and activated HSCs was analyzed by western blotting. Immunoprecipitation was used to assess the interaction of ASCT2 and O-GlcNAc transferase (OGT). In addition, ASCT2 protein stability was assayed after cycloheximide (CHX) treatment. The O-GlcNAcylation site of ASCT2 was predicted and mutated by site-directed mutagenesis. Real-time PCR, immunofluorescence, kit determinations and Seahorse assays were used to clarify the effect of ASCT2 O-GlcNAcylation on HSC glutaminolysis and HSC activation. Western blotting, immunochemistry, and immunohistofluorescence were used to analyze the effect of ASCT2 O-GlcNAcylation in vivo.

Results

We observed significantly increased O-GlcNAcylation modification of ASCT2. ASCT2 was found to interact with OGT to regulate ASCT2 stability. We predicted and confirmed that O-GlcNAcylation of ASCT2 at Thr122 site resulted in HSCs activation. We found Thr122 O-GlcNAcylation of ASCT2 mediated membrane trafficking of glutamine transport and attenuated HSC glutaminolysis. Finally, we validated the expression and function of ASCT2 O-GlcNAcylation after injection of AAV8-ASCT2 shRNA in CCl₄-induced liver fibrosis mice in vivo.

Conclusions

Thr122 O-GlcNAcylation regulation of ASCT2 resulted in stability and membrane trafficking-mediated glutaminolysis in HSCs and liver fibrosis. Further studies are required to assess its role as a putative therapeutic target.

SUBMITTER: Wang F

PROVIDER: S-EPMC9634767 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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<i>O</i>-GlcNAcylation Coordinates Glutaminolysis by Regulating the Stability and Membrane Trafficking of ASCT2 in Hepatic Stellate Cells.

Wang Feixia F Chen Li L Zhang Biyun B Li Zhanghao Z Shen Min M Tang Li L Zhang Zili Z Shao Jiangjuan J Zhang Feng F Zheng Shizhong S Tan Shanzhong S

Journal of clinical and translational hepatology 20220311 6

<h4>Background and aims</h4>Recognition of excessive activation of hepatic stellate cells (HSCs) in liver fibrosis prompted us to investigate the regulatory mechanisms of HSCs. We aimed to examine the role of O-GlcNAcylation modification of alanine, serine, cysteine transporter 2 (ASCT2) in HSCs and liver fibrosis.<h4>Methods</h4>The expression of O-GlcNAcylation modification in fibrotic mice livers and activated HSCs was analyzed by western blotting. Immunoprecipitation was used to assess the i ...[more]

PMID: 36381090

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