Establishment and Characterization of Hepatocyte Line from Turbot (Scophthalmus maximus L.) and Its Application in the Study of Glucose Metabolism

Abstract

The present study was to establish a hepatocyte line and investigate its role in glucose metabolism. A continuous cell line, THL (turbot hepatocyte line), was established from the liver tissue of turbot (Scophthalmus maximus L.). It has been successfully passaged more than 60 generations. The THL cells showed an epithelial-like morphology and the normal chromosome number was 44. Different methods were used to identify the hepatocytes. Periodic acid-Schiff (PAS) staining for THL cells was positive, and two key functional proteins of hepatocytes, cytokeratin- 18 (CK- 18) and albumin (ALB), were detected in THL cells. The results of CCK- 8 indicated that a medium containing 15 mM glucose showed optimal cell viability of THL. Conversely, elevating glucose concentrations beyond 50 mM markedly impaired THL cell viability. Western blot and qRT-PCR were employed to assess the gene and protein expression in cells treated with varying concentrations of glucose. The results of cells incubated with 0 mM, 15 mM, and 50 mM glucose concentrations showed that compared with the 0 mM glucose group, 15 mM glucose could increase the gene expression of glucokinase (gk) and decrease the gene expression of cytosolic phosphoenolpyruvate carboxykinase (cpepck), mitochondrial phosphoenolpyruvate (mpepck), glucose- 6-phosphatase 1 (g6pase1), forkhead box o1 (foxo1), and glucose-regulated protein 78 (grp78). Compared to 15 mM glucose treatment, the expression of gk in the 50 mM group was significantly decreased, but the expression of cpepck, mpepck, g6pase1, foxo1, and grp78 was significantly increased. Moreover, the protein expression of FoxO1 and GRP78 in 50 mM treatment group was significantly increased compared to that in the15 mM group. In the present study, it was found that excessive glucose level can activate the pathways of FoxO1-mediated gluconeogenesis and GRP78-mediated endoplasmic reticulum stress and reduce the glycolytic pathway, thus disrupting the glucose homeostasis in hepatocytes.