Figure 2. E4orf1 is ‘sufficient’ to up-regulate cellular glucose uptake and to up-regulate the Ras/PI3k pathway.
3T3-E4(pTRE E4orf1) and pTRE Null (pTRE) cell lines were treated with varying doses of Doxycycline for 24 h, and a dose-dependent 2DG uptake was determined. Protein was also harvested from 3T3-E4 and pTRE Null treated with 1,000 ng/ml Doxycycline for 24 hours to determine signaling changes with E4orf1 expression. A) E4orf1 is sufficient to induce a dose-response increase in glucose uptake. 3T3-E4 and pTRE Null control cell lines were treated with 500, 750, or 1,000 ng/ml Doxycycline to induce different levels of E4orf1 expression, 2DG uptake was determined. Compared to pTRE, the E4orf1 expressing cell had significantly greater glucose uptake when treated with 750 and 1,000 ng/ml Doxycycline (p * = 0.007 and ** = 2.6×10−7, respectively). B) Ras WB and densitometry normalized to β-actin for 3T3-E4 and pTRE Null treated with 1,000 ng/ml Doxycycline for 24 hours. Ras expression was significantly higher in the 3T3-E4 cell line (p = 0.05). C) p-AKT and AKT WB and densitometry expressed as ratio of p-AKT to total AKT in 3T3-E4 and pTRE Null treated with 1,000 ng/ml Doxycycline for 24 hours. p-AKT is significantly higher in the 3T3-E4 cell line (p = 3.54*10−5). D) Glut4 WB and densitometry normalized to β-actin for 3T3-E4 and pTRE Null treated with 1,000 ng/ml Doxycycline for 24 hours. Glut4 expression was significantly higher in the 3T3-E4 cell line (p = 0.05). E) Glut1 WB and densitometry normalized to β-actin for 3T3-E4 and pTRE Null treated with 1,000 ng/ml Doxycycline for 24 hours. Glut1 expression was significantly higher in the 3T3-E4 cell line (p = 0.01). F) Adiponectin WB and densitometry normalized to β-actin for 3T3-E4 and pTRE Null treated with 1,000 ng/ml Doxycycline for 24 hours. Adiponectin expression is significantly higher in the 3T3-E4 cell line (p = 0.006).