As the main detoxification organ that is routinely exposed to environmental insults, hepatocytes of the adult liver retain the ability to re-enter the cell cycle and restore the original mass upon injury and tissue loss. However, massive injury can lead to liver failure, and chronic injury can cause liver fibrosis, cirrhosis, and hepatocellular carcinoma. For example, acetaminophen overdose can lead to hepatic oxidative injury by glutathione depletion. Currently, the genetic regulation of organ repair is only partially understood, and novel insights will aid the development of innovative treatments for liver disease. The mouse model of hereditary tyrosinemia type I (HTI), an inborn error of tyrosine metabolism caused by the loss of fumarylacetoacetate hydrolase (FAH), provides an excellent system to study liver regeneration. FAH deficiency leads to toxic metabolite accumulation and hepatocyte death. The drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3- cyclohexanedione (NTBC) prevents hepatocyte death in both HTI patients and Fah-/- mice by inhibiting an upstream metabolic enzyme, which blocks toxin formation. I previously used the Fah-/- model combined with translating ribosome affinity purification (TRAP) to obtain RNA from repopulating hepatocytes. High-throughput sequencing (TRAP-seq) identified multiple genes involved in GSH metabolism as the most highly induced. In particular, the top hit was Slc7a11, which encodes the cystine/glutamate antiporter (xCT), imports cystine for glutathione synthesis to defend against oxidative stress. Therefore, I hypothesize that xCT upstream of GSH synthesis plays a central role in redox regulation in repopulating hepatocytes.
In Aim 1, I will test the function of Slc7a11 during liver regeneration. I will employ cDNA and shRNA constructs targeting Slc7a11 to perform overexpression and loss-of-function studies, respectively, to investigate the degree to which xCT controls liver regeneration following toxic injury. I will also administer erastin, an xCT inhibitor, to Fah-/- mice and determine its effects on liver repopulation.
In Aim 2, I propose to establish an in vivo model that allows for quantitative detection and spatiotemporal mapping of redox dynamics during liver regeneration. I will utilize the Fah-/- model and integrate a ratiometric GFP biosensor specific to glutathione redox potential (Grx1-RoGFP) to detect cytoplasmic glutathione redox potential changes during liver repopulation. This study will address the question of whether replicating hepatocytes have higher or lower redox states relative to quiescent hepatocytes. In addition, I will perturb the level of expression of Slc7a11 and to assess the effect on the liver?s redox state. This study will provide an in-depth understanding of temporal and regional specificity of redox regulation during liver repopulation and will identify novel potential targets that might be employed for the treatment of acute liver injury by inducing hepatocyte replication.

Public Health Relevance

Understanding the regulation of liver regeneration will help the development of novel treatments for liver disease. With the integration of cell-type-specific expression profiling of repopulating hepatocytes and quantitative in vivo mapping of redox status, this study will establish the control of the oxidation/reduction response during liver injury and identify therapeutic targets that promote hepatocyte recovery.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31DK113666-01A1
Application #
9468949
Study Section
Special Emphasis Panel (ZDK1)
Program Officer
Densmore, Christine L
Project Start
2017-09-01
Project End
2020-08-31
Budget Start
2017-09-01
Budget End
2018-08-31
Support Year
1
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Wang, Amber W; Wangensteen, Kirk J; Wang, Yue J et al. (2018) TRAP-seq identifies cystine/glutamate antiporter as a driver of recovery from liver injury. J Clin Invest 128:2297-2309