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Generation of carbamoyl phosphate synthetase 1 reporter cell lines for the assessment of ammonia metabolism.
- Source :
-
Journal of cellular and molecular medicine [J Cell Mol Med] 2017 Dec; Vol. 21 (12), pp. 3214-3223. Date of Electronic Publication: 2017 May 30. - Publication Year :
- 2017
-
Abstract
- Both primary hepatocytes and stem cells-derived hepatocyte-like cells (HLCs) are major sources for bioartificial liver (BAL). Maintenance of hepatocellular functions and induction of functional maturity of HLCs are critical for BAL's support effect. It remains difficult to assess and improve detoxification functions inherent to hepatocytes, including ammonia clearance. Here, we aim to assess ammonia metabolism and identify ammonia detoxification enhancer by developing an imaging strategy. In hepatoma cell line HepG2, and immortalized hepatic cell line LO2, carbamoyl phosphate synthetase 1 (CPS1) gene, the first enzyme of ammonia-eliminating urea cycle, was labelled with fluorescence protein via CRISPR/Cas9 system. With the reporter-based screening approach, cellular detoxification enhancers were selected among a collection of 182 small molecules. In both CPS1 reporter cell lines, the fluorescence intensity is positively correlated with cellular CPS1 mRNA expression, ammonia elimination and secreted urea, and reflected ammonia detoxification in a dose-dependent manner. Surprisingly, high-level CPS1 reporter clones also reserved many other critical hepatocellular functions, for example albumin secretion and cytochrome 450 metabolic functions. Sodium phenylbutyrate and resveratrol were identified to enhance metabolism-related gene expression and liver-enriched transcription factors C/EBPα, HNF4α. In conclusion, the CPS1-reporter system provides an economic and effective platform for assessment of cellular metabolic function and high-throughput identification of chemical compounds that improve detoxification activities in hepatic lineage cells.<br /> (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)
- Subjects :
- Albumins metabolism
CCAAT-Enhancer-Binding Proteins genetics
CCAAT-Enhancer-Binding Proteins metabolism
CRISPR-Cas Systems
Carbamoyl-Phosphate Synthase (Ammonia) metabolism
Cell Line, Transformed
Cytochrome P-450 Enzyme System genetics
Cytochrome P-450 Enzyme System metabolism
Gene Editing
Gene Expression Regulation drug effects
Genes, Reporter
Hep G2 Cells
Hepatocyte Nuclear Factor 4 genetics
Hepatocyte Nuclear Factor 4 metabolism
Humans
Liver, Artificial
Luminescent Proteins genetics
Luminescent Proteins metabolism
Phenylbutyrates pharmacology
Resveratrol
Small Molecule Libraries pharmacology
Stilbenes pharmacology
Urea metabolism
Red Fluorescent Protein
Ammonia metabolism
Carbamoyl-Phosphate Synthase (Ammonia) genetics
Founder Effect
High-Throughput Screening Assays
Inactivation, Metabolic genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1582-4934
- Volume :
- 21
- Issue :
- 12
- Database :
- MEDLINE
- Journal :
- Journal of cellular and molecular medicine
- Publication Type :
- Academic Journal
- Accession number :
- 28557353
- Full Text :
- https://doi.org/10.1111/jcmm.13225