E4orf1: A Novel Ligand That Improves Glucose Disposal in Cell Culture

Reducing dietary fat intake and excess adiposity, the cornerstones of behavioral treatment of insulin resistance(IR), are marginally successful over the long term. Ad36, a human adenovirus, offers a template to improve IR, independent of dietary fat intake or adiposity. Ad36 increases cellular glucose uptake via a Ras-mediated activation of phosphatidyl inositol 3-kinase(PI3K), and improves hyperglycemia in mice, despite a high-fat diet and without reducing adiposity. Ex-vivo studies suggest that Ad36 improves hyperglycemia in mice by increasing glucose uptake by adipose tissue and skeletal muscle, and by reducing hepatic glucose output. It is impractical to use Ad36 for therapeutic action. Instead, we investigated if the E4orf1 protein of Ad36, mediates its anti-hyperglycemic action. Such a candidate protein may offer an attractive template for therapeutic development. Experiment-1 determined that Ad36 ‘requires’ E4orf1 protein to up-regulate cellular glucose uptake. Ad36 significantly increased glucose uptake in 3T3-L1 preadipocytes, which was abrogated by knocking down E4orf1 with siRNA. Experiment-2 identified E4orf1 as ‘sufficient’ to up-regulate glucose uptake. 3T3-L1 cells that inducibly express E4orf1, increased glucose uptake in an induction-dependent manner, compared to null vector control cells. E4orf1 up-regulated PI3K pathway and increased abundance of Ras–the obligatory molecule in Ad36-induced glucose uptake. Experiment-3: Signaling studies of cells transiently transfected with E4orf1 or a null vector, revealed that E4orf1 may activate Ras/PI3K pathway by binding to Drosophila discs-large(Dlg1) protein. E4orf1 activated total Ras and, particularly the H-Ras isoform. By mutating the PDZ domain binding motif(PBM) of E4orf1, Experiment-4 showed that E4orf1 requires its PBM to increase Ras activation or glucose uptake. Experiment-5: In-vitro, a transient transfection by E4orf1 significantly increased glucose uptake in preadipocytes, adipocytes, or myoblasts, and reduced glucose output by hepatocytes. Thus, the highly attractive anti-hyperglycemic effect of Ad36 is mirrored by E4orf1 protein, which may offer a novel ligand to develop anti-hyperglycemic drugs

Doxycycline-regulated 3T3-L1 preadipocyte cell line with inducible, stable expression of adenoviral E4orf1 gene: a cell model to study insulin-independent glucose disposal.

Impaired glycemic control and excessive adiposity are major risk factors for Type 2 Diabetes mellitus. In rodent models, Ad36, a human adenovirus, improves glycemic control, independent of dietary fat intake or adiposity. It is impractical to use Ad36 for therapeutic action. Instead, we identified that E4orf1 protein of Ad36, mediates its anti-hyperglycemic action independent of insulin signaling. To further evaluate the therapeutic potential of E4orf1 to improve glycemic control, we established a stable 3T3-L1 cell system in which E4orf1 expression can be regulated. The development and characterization of this cell line is described here. Full-length adenoviral-36 E4orf1 cDNA obtained by PCR was cloned into a tetracycline responsive element containing vector (pTRE-Tight-E4orf1). Upon screening dozens of pTRE-Tight-E4orf1 clones, we identified the one with the highest expression of E4orf1 in response to doxycycline treatment. Furthermore, using this inducible system we characterized the ability of E4orf1 to improve glucose disposal in a time dependent manner. This stable cell line offers a valuable resource to carefully study the novel signaling pathways E4orf1 uses to enhance cellular glucose disposal independent of insulin

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 improves lipid and glucose metabolism in hepatocytes: a template to improve steatosis & hyperglycemia

Hepatic steatosis often accompanies obesity and insulin resistance. The cornerstones of steatosis treatment include reducing body weight and dietary fat intake, which are marginally successful over the long term. Ad36, a human adenovirus, may offer a template to overcome these limitations. In vitro and in vivo studies collectively indicate that via its E4orf1 protein, Ad36 improves hyperglycemia, and attenuates hepatic steatosis, despite a high fat diet and without weight loss. Considering that hepatic insulin sensitivity, or the synthesis, oxidation, or export of fatty acid by hepatocytes are the key determinant of hepatic lipid storage, we determined the role of E4orf1 protein in modulating these physiological pathways. For this study, HepG2 cells, or mouse primary hepatocytes were transfected with E4orf1 or the null vector. Glucose output by hepatocytes was determined under gluconeogenic conditions (cAMP and dexamethasone, or glucagon exposure). Also, de-novo lipogenesis, palmitate oxidation, and lipid export as determined by apoB secretion were measured 48 h post transfection. Results show that compared to null vector transfected cells, E4orf1 significantly reduced glucose output in basal and gluconeogenic conditions. E4orf1 reduced de-novo lipogenesis by about 35%, increased complete fatty acid oxidation 2-fold (p\u3c0.0001), and apoB secretion 1.5 fold(p\u3c0.003). Response of key signaling molecules to E4orf1 transfection was in agreement with these findings. Thus, E4orf1 offers a valuable template to exogenously modulate hepatic glucose and lipid metabolism. Elucidating the underlying molecular mechanism may help develop therapeutic approaches for treating diabetes or non-alcoholic fatty liver disease(NAFLD)

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

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