Stable expression of 3T3-L1/pTet-On and pTRE-Tight-E4orf1 plasmids and E4orf protein expression.

<p><b>A</b>) Flourescence micrographs (40×) of live <i>E4orf1</i> double stable inducible clone induced with 1,000 ng/mL doxycycline for 24 h. Phase, p-Tet-On showing green fluorescence, p-TRE-Tight-E4orf1-Red fluorescence and Overlay. Arrows indicate co-localization. B) Western blot showing E4orf1 protein expression in pTRE and 3T3-E4 clone.</p

Transient transfection of 3T3-L1/Tet-On clone by pTRE-Tight-Luc and luciferase assay.

<p>3T3-L1-pTet-On clone was transfected with pTRE-Tight-Luc plasmid. After 48 h post transfection, cells were treated with 0, 100, 1,000 ng/mL or 10,000 ng/mL of Dox and 6 h, 12 h and 48 h post treatment, the luciferase activity was analyzed using the luciferase assay system kit.</p

E4orf1 fold expression relative to pTRE.

<p>The pTRE TIGHT-E4orf1 clone was treated with 0 and 1,000 ng/mL Dox and RNA was harvested at either 16, 24 or 48 h post treatment. E4orf1 fold expression was determined relative to un-induced pTRE TIGHT-E4orf1 at 24 h using real time PCR assay.</p

Effect of regulated <i>E4orf1</i> expression on glucose disposal.

<p>3T3-E4 inducible clone and pTRE empty vector clones were induced with 1,000 ng/mL Dox for 24, 48, 72 and 96 h. After 96 h of induction, Dox was removed for 24 h or 48 h and reintroduced for 24 h or 48 h. At all-time points the glucose uptake in E4orf1 groups was significantly greater compared to the respective pTRE groups as determined by student T-Test (p<0.00001). Mean + SD. The difference of glucose uptake between pTRE and E4orf1 groups is presented, which was calculated by subtracting the average of the pTRE group values from the individual biological replicate value for the respective E4orf1 group.</p

E4orf1 requires its PBM for glucose uptake and Ras activation.

<p>3T3-L1 cells were transfected with V-5 tagged plasmids expressing E4orf1 (pcDNA-V5-AD36-E4orf1), mutant E4orf1 (pcDNA-V5- AD36-E4ORF1ΔPBM) or a null vector (pcDNA-V5-DEST) and a Ras activation assay was conducted the next day. The activated slurry and 3% of whole cell lystate was loaded to SDS-PAGE and probed for Ras. Also 3T3-L1 cells that constitutively express Ad36 E4orf1, a mutated E4orf1 (deleted PBM; ΔPBM) or Null vector were plated in 12 well plates and a 2DG uptake assay was conducted the following day. <b>A</b>) WB for Ras activation assay: pcDNA-V5-AD36-E4orf1 activates Ras (p = 0.04), whereas the Null vector (pcDNA-V5-DEST) does not. pcDNA-V5- AD36-E4ORF1ΔPBM did not activate Ras, suggesting E4orf1 depends on its PBM region for Ras activation. Densitometry expressed as ratio of activated Ras to normalized total Ras from 3% WCL WB. <b>B</b>) 2DG uptake in constitutive expressing Ad36 E4orf1, a mutated E4orf1 (deleted PBM; ΔPBM) or Null vector normalized to protein: E4orf1 increased glucose uptake compared to Null vector (p = 0.008), and there was no difference in glucose uptake when the PBM region of E4orf1 is mutated.</p

E4orf1 interacts with Dlg1 and activates Ras.

<p>HEK293 were co-transfected with GW1-CMV-HA-Dlg1 and either pcDNA-V5-Ad36 E4orf1 or a control plasmid (pcDNA-V5-DEST), and a pull down for HA was conducted and loaded to SDS-PAGE. WB were probed with V5 antibody to detect Dlg1 and E4orf1 interaction. Also, 3T3-E4 and pTRE Null cell lines were treated with 1,000 ng/ml Doxycycline, and a Ras activation assay was conducted. Following pull down, activated slurry and 6% of the whole cell lysate were loaded to SDS-PAGE and probed with total Ras antibodies, or H-, N-, or K-Ras specific antibodies. Blots were also probed with β-actin, and densitometry was expressed as ratio of activated Ras to normalized total Ras. <b>A</b>) WB show HA-Dlg1 and V5E4orf1 in the same complex after IP, indicating the binding of Ad36 E4orf1 with Dlg1. WB also shows the presence of Dlg1 in the whole cell lysate (WCL). As expected, V5-E4orf1 is absent in pcDNA-V5-DEST group after IP for HA-Dlg1. <b>B</b>. (i) WB for total, H-, and N-Ras activation. T = total (6% WCL) and A = Activated slurry. (ii) Total Ras abundance normalized to β-actin was significantly more in 3T3-E4 compared to pTRE Null vector cell line (p = 0.04). (iii)Total Ras activation densitometry, expressed as ratio of activated to normalized total Ras. Total Ras activation was significantly greater in 3T3-E4 compared to pTRE Null (p = 0.01). (iv) H-ras activation densitometry, expressed as ratio of activated H-Ras to normalized total H-Ras. H-Ras activation was significantly more in 3T3-E4 compared to pTRE Null (p = 0.01).</p

E4orf1 is ‘sufficient’ to up-regulate cellular glucose uptake and to up-regulate the Ras/PI3k pathway.

<p>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. <b>A</b>) 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>B</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). <b>C</b>) 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). <b>D</b>) 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). <b>E</b>) 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). <b>F</b>) 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).</p

DNL in HepG2 following E4orf1 transfection.

<p>HepG2 cells were transfected with E4orf1 or null vector. A) Expressed as mean fold change in pmol [¹⁴-C]-glucose/mg protein relative to null, ± SD. Two separate experiments were combined. E4orf1 significantly decreases DNL compared to null (p = 0.01). B) E4orf1 significantly decreases phosphorylated SREBP1c expression compared to null (V5) (p = 0.001) indicating reduced DNL in these cells.</p

Glucose Output from Primary Hepatocytes Transfected with E4orf1.

<p>Glucose output in mouse primary hepatocytes after transfection with E4orf1 or null vector. A) Glucose output by primary hepatocytes after serum and glucose starvation for 1.5 hours, expressed as mean mM glucose per mg protein ± SD. E4orf1suppressed glucose output compared to Null vector (p = 0.0001). B) Glucose output in primary hepatocytes treated with 100 nM glucagon in serum-free, and glucose-free media for 1.5 hr. E4orf1 suppressed glucose output in this condition (p = 0.029). Expressed as mean mM glucose per mg protein, ± SD.</p

E4orf1 suppresses glucose output in the presence of gluconeogenic stimulators in HepG2.

<p>(A) Western blot showing Ad36 E4orf1 protein expression in HepG2 cells following transfection with V5-Ad36 E4orf1 plasmid DNA. (B) Glucose output by HepG2 cells following E4orf1 or null Vector transfection. Cells were stimulated with gluconeogenic cAMP (1 mM) (C) and Dexamethasone (500 nM) (D) in the absence or presence of insulin (I). Expressed as mean mM glucose per mg of protein, ± SD. E4orf1 significantly suppresses glucose output in the basal and insulin stimulated conditions (p = 0.0001 and 0.003, respectively).</p