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

Feixia Wang; Li Chen; Biyun Zhang; Zhanghao Li; Min Shen; Li Tang; Zili Zhang; Jiangjuan Shao; Feng Zhang; Shizhong Zheng; Shanzhong Tan

doi:10.14218/JCTH.2021.00413

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

J Clin Transl Hepatol. 2022 Dec 28;10(6):1107-1116. doi: 10.14218/JCTH.2021.00413. Epub 2022 Mar 11.

Authors

Feixia Wang^{1

2}, Li Chen², Biyun Zhang³, Zhanghao Li², Min Shen², Li Tang^{1

2}, Zili Zhang², Jiangjuan Shao², Feng Zhang², Shizhong Zheng², Shanzhong Tan¹

Affiliations

¹ Department of Integrated TCM and Western Medicine, Nanjing Hospital Affiliated to Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.
² Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.
³ Department of Nuclear Medicine, Jiangsu Province Hospital of Chinese Medicine, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.

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.

Keywords: ASCT2; Liver fibrosis; O-GlcNacylation; Stability; Trafficking.