Skip to main content
. 2019 Sep 3;10:605. doi: 10.3389/fendo.2019.00605

Figure 13.

Figure 13

Function role of HNF1α and CREB in glucagon-induced IGF-I promoter activity in αT3 cells. (A) Mapping of glucagon responsive sequence to the proximal promoter of IGF-I gene with CRE and HBE sites. 5′ Deletion was conducted in the 1,077 bp promoter of pIGF1(-1077).Luc to generate a series of 5′ deleted Luc reporters with decreasing lengths of IGF-I promoter from position −874 to −44. These 5′ deletion constructs were then used for transfection study in αT3 cells followed by 24 h treatment with glucagon (100 nM). Over-expression of (B) HNF1α and (C) CREB on IGF-I promoter activation. Co-transfection was performed in αT3 cells with pIGF1(-112).Luc (with CRE and HBE sites in proximal promoter) together with increasing levels of the expression vector for carp HNF1α or CREB as indicated. (D) Functional interaction of HNF1α and CREB on glucagon-induced IGF-I promoter activity expressed in αT3 cells. In parallel study with pIGF1(-112).Luc, similar transfection was conducted with the HNF1α vector alone (30 ng/ml), CREB vector alone (30 ng/ml) or co-transfection with the two vectors (30 ng/well each). After that, the cells were treated with glucagon (100 nM) for 24 h. In our experiments with IGF-I promoter, cell lysate was prepared from αT3 cells after the drug treatment/transfection with expression vectors and used for luciferase activity measurement. (E) Protein:protein interaction of HNF1α and CREB induced by glucagon in carp hepatocytes. Immunoprecipitation (IP) was performed in cell lysate prepared from carp hepatocytes after 1-h treatment with glucagon (10 nM) using the antibody for HNF1α. After that, the protein pull-down was subjected to immunoblotting (IB) with antibodies for phosphorylated and total protein of CREB (as “P-CREB” and “T-CREB,” respectively). In this experiment, parallel IP with the antibody for mouse IgG (as “anti-IgG”) was used as the negative control while the corresponding IB for HNF1α, T-CREBk, and β actin were conducted in the cell lysate prior to the pull-down with HNF1α antibody to serve as the input control.