The G6pc1 Knockout H-4-II-E Cell Line is a CRISPR/Cas9-edited knockout cell line derived from the rat hepatoma H-4-II-E cell line, designed to disrupt the G6pc1 gene encoding the catalytic subunit of glucose-6-phosphatase. This loss-of-function model enables detailed investigation of hepatic glucose-6-phosphate hydrolysis, a pivotal reaction in gluconeogenesis and glycogenolysis. CRISPR/Cas9-mediated gene disruption creates a stable knockout background for studying G6pc1-dependent metabolic processes in a hepatocyte-like environment.
H-4-II-E is an adherent hepatic epithelial cell line originally established from a Reuber hepatoma in an ACI rat. This cell line retains key characteristics of differentiated hepatocytes, including responsiveness to hormonal stimuli and expression of liver-specific metabolic enzymes, making it a widely accepted model hepatocyte line for studying hepatic metabolism, insulin signaling, and related pathologies. Its rat origin provides a relevant platform for translational research into liver function and disease.
G6pc1 encodes glucose-6-phosphatase, an endoplasmic reticulum?Cresident enzyme that catalyzes the hydrolysis of glucose-6-phosphate to free glucose and inorganic phosphate. This reaction is rate-limiting for hepatic glucose output and is essential for maintaining blood glucose homeostasis during fasting. G6PC1 function is intimately coupled with the glucose-6-phosphate transporter SLC37A4, which provides substrate inside the ER. G6pc1 expression is transcriptionally regulated by key metabolic transcription factors including FOXO1, CREB, and HNF4??, downstream of glucagon-stimulated cAMP/PKA signaling, and is suppressed by insulin through the PI3K/Akt pathway. Additional modulators include dexamethasone and cAMP, which further integrate nutritional and hormonal cues. The glucose liberated by G6PC1 is exported via the glucose transporter SLC2A2 (GLUT2), completing the gluconeogenic/glycogenolytic pathway that also involves PCK1, FBP1, and GCK.
In the H-4-II-E hepatoma context, knockout of G6pc1 effectively abrogates endogenous glucose-6-phosphatase activity, providing a clinically relevant model for glycogen storage disease type Ia (von Gierke disease), which results from G6PC1 deficiency and manifests as hypoglycemia, hyperlipidemia, and hepatocellular carcinoma predisposition. This knockout cell line permits dissection of G6PC1-dependent versus -independent effects on metabolic flux, insulin sensitivity, and lipid metabolism in a simplified yet physiologically recognizable hepatic setting. Moreover, it facilitates examination of compensatory pathways and off-target effects of therapeutic interventions aimed at restoring glucose homeostasis.
Researchers can employ this knockout model in a variety of experimental paradigms, including Western blotting and RT-qPCR to confirm gene disruption, glucose-6-phosphatase enzymatic activity assays, and colorimetric/fluorometric glucose output measurements. The cell line is suitable for high-throughput screening of small molecules that modulate hepatic glucose production, metabolic flux analyses using Seahorse technology, and insulin signaling studies via phospho-Akt analysis. Transcriptomic profiling by RNA-seq can reveal global gene expression changes upon G6pc1 loss. These applications support investigations into hepatic gluconeogenesis, insulin resistance, diabetes, and glycogen storage disease pathogenesis. For further information, please contact Ascent Research.
