PXR activation impairs hepatic glucose metabolism partly via inhibiting the HNF4α-GLUT2 pathway

Drug-induced hyperglycemia/diabetes is a global issue. Some drugs induce hyperglycemia by activating the pregnane X receptor (PXR), but the mechanism is unclear. Here, we report that PXR activation induces hyperglycemia by impairing hepatic glucose metabolism due to inhibition of the hepatocyte nuclear factor 4-alpha (HNF4α)‒glucose transporter 2 (GLUT2) pathway. The PXR agonists atorvastatin and rifampicin significantly downregulated GLUT2 and HNF4α expression, and impaired glucose uptake and utilization in HepG2 cells. Overexpression of PXR downregulated GLUT2 and HNF4α expression, while silencing PXR upregulated HNF4α and GLUT2 expression. Silencing HNF4α decreased GLUT2 expression, while overexpressing HNF4α increased GLUT2 expression and glucose uptake. Silencing PXR or overexpressing HNF4α reversed the atorvastatin-induced decrease in GLUT2 expression and glucose uptake. In human primary hepatocytes, atorvastatin downregulated GLUT2 and HNF4α mRNA expression, which could be attenuated by silencing PXR. Silencing HNF4α downregulated GLUT2 mRNA expression. These findings were reproduced with mouse primary hepatocytes. Hnf4α plasmid increased Slc2a2 promoter activity. Hnf4α silencing or pregnenolone-16α-carbonitrile (PCN) suppressed the Slc2a2 promoter activity by decreasing HNF4α recruitment to the Slc2a2 promoter. Liver-specific Hnf4α deletion and PCN impaired glucose tolerance and hepatic glucose uptake, and decreased the expression of hepatic HNF4α and GLUT2. In conclusion, PXR activation impaired hepatic glucose metabolism partly by inhibiting the HNF4α‒GLUT2 pathway. These results highlight the molecular mechanisms by which PXR activators induce hyperglycemia/diabetes.

Hepatocyte Nuclear Factor 4α Inhibits Expression of Vascular Endothelial Growth Factor in Human Hepatocellular Carcinoma Cell Lines and Tube Formation of Human Umbilical Vein Endothelial Cell / 胃肠病学

Background: Hepatocyte nuclear factor 4α (HNF4α) plays an important role in the development of liver,and studies demonstrate that it is correlated with the pathogenesis of hepatocellular carcinoma (HCC).However,the regulatory effect of HNF4α on expression of vascular endothelial growth factor (VEGF) in human HCC cell lines and tube formation of human umbilical vein endothelial cell (HUVEC) is not yet clear.Aims: To investigate the effect of HNF4α on expression of VEGF in human HCC cell lines and tube formation of HUVEC.Methods: Lentiviral vector overexpressed HNF4α was constructed,and then transfected into HepG2 and SMMC-7721 cells (experimental group),cells transfected with lentiviral blank vector and cells without transfection were served as negative control group and blank control group,respectively.The mRNA and protein expressions of HNF4α,VEGF were detected by qRT-PCR and Western blotting,respectively.The conditioned media of HepG2 and SMMC-7721 cells were co-cultured with HUVEC,and number of HUVEC tube formation was measured.Results: HepG2 and SMMC-7721 cells with stable overexpression of HNF4α were successfully established.Compared with negative control group and blank control group,mRNA and protein expressions of VEGF in experimental group were significantly decreased (P＜0.05),and number of HUVEC tube formation was significantly decreased (P＜0.05).Conclusions: HNF4α can significantly inhibit the expression of VEGF in HepG2 and SMMC-7721 cells and tube formation of HUVEC.

Inhibitory effect of PEP-1-mediated recombinant hepatocyte nuclear factor 4 alpha transduction on hepatocellular carcinoma cells / 第二军医大学学报

Objective To investigate cell penetrating peptide (PEP-1)-mediated transduction of recombinant hepatocyte nuclear factor 4 alpha (HNF4α) protein into hepatocellular carcinoma (HCC) cells, and to observe the effect of the fusion protein P-HNF4α on HCC cells. Methods The expression vector pET28a-P-HNF4α was constructed. The prokaryotic expression condition of fusion protein P-HNF4α was optimized. Recombinant P-HNF4α carrying cell penetrating peptide PEP-1 was obtained by abundant expression, purified by affinity chromatography, and was concentrated and dialyzed. P-HNF4α was transduced into HCC cells. The transduction efficiency was analyzed by Western blotting analysis. Sub-cellular localization of P-HNF4α was detected by Western blotting analysis with nuclear and cytoplasmic extracts and confirmed by immunofluorescence assay. Real-time RT-PCR was used to examine the gene expression of HCC cells. The proliferation of HCC cells was detected with CCK-8 kit. The migration and invasion of HCC cells were detected by wound-healing assay and trans-well invasion assay, respectively. Results P-HNF4α was efficiently transduced into Huh7 cells and located in the nucleus as mediated by PEP-1. P-HNF4α significantly up-regulated the expression of characteristic hepatocyte markers and down-regulated the "stemness" genes in Huh7 cells (P<0.05 or P<0.01). Moreover, the proliferation (P<0.05), migration (P<0.001) and invasion (P<0.05) of HCC cells were significantly suppressed by fusion protein P-HNF4α. Conclusion P-HNF4α can induce the differentiation of HCC cells to mature hepatocytes and reduce the malignancy phenotype of HCC cells, suggesting that PEP-1-mediated HNF4α protein transduction may be a potential strategy for HCC differentiation therapy.