An adenovirus-derived protein: A novel candidate for anti-diabetic drug development

Abstract

Aims: Exposure to human adenovirus Ad36 is causatively and correlatively linked with better glycemic control in animals and humans, respectively. Although the anti-hyperglycemic property of Ad36 may offer some therapeutic potential, it is impractical to use an infectious agent for therapeutic benefit. Cell-based studies identified that Ad36 enhances cellular glucose disposal via its E4orf1 protein. Ability to improve glycemic control in vivo is a critical prerequisite for further investigating the therapeutic potential of E4orf1. Therefore, the aim of this study was to determine the ability of E4orf1 to improve glycemic control independent of insulin despite high fat diet.

Materials & methods: 8-9wk old male C57BL/6J mice fed a high-fat diet (60% kcal) were injected with a retrovirus plasmid expressing E4orf1, or a null vector (Control). Glycemic control was determined by glucose and insulin tolerance test. Islet cell size, amount of insulin and glucagon were determined in formalin-fixed pancreas. Rat insulinoma cell line (832/13) was infected with E4orf1 or control to determine changes in glucose stimulated insulin secretion. Protein from flash frozen adipose tissue depots, liver and muscle was used to determine molecular signaling by western blotting.

Results: In multiple experiments, retrovirus-mediated E4orf1 expression in C57BL/6J mice significantly and reproducibly improved glucose excursion following a glucose load despite a high fat diet (60% energy). Importantly, E4orf1 improved glucose clearance without increasing insulin sensitivity, production or secretion, underscoring its insulin-independent effect. E4orf1 modulated molecular signaling in mice tissue, which included greater protein abundance of adiponectin, p-AKT and Glucose transporter Glu4.

Conclusions: This study provides the proof of concept for translational development of E4orf1 as a potential anti-diabetic agent. High fat intake and impaired insulin signaling are often associated with obesity, diabetes and insulin resistance. Hence, the ability of E4orf1 to improve glycemic control despite high fat diet and independent of insulin, is particularly attractive.

Keywords: Adenoviral protein; Anti-diabetic drug; Diabetes; Glucose disposal; Glycemic control; Insulin-independent effect.

Fig. 1

E4orf1 enhances blood glucose excursion in high fat fed mice: A. Eighteen, weight-matched 7 week old C57Bl/6J male mice on chow diet were divided into two groups (Control; n = 9 and E4orf1; n = 9) followed by baseline glucose tolerance test (GTT; black solid line). The mice were fed a 60% kcal high fat (HF) diet for 2 week and GTT performed (black dotted line). The animals were inoculated with pBabe retrovirus expressing control puro (B–C) or Ad36 E4orf1 (D–E) and GTT performed 1 week post inoculation. (*p < 0.05, **p < 0.005, ***p < 0.0005). The bar graphs show area under the curve for respective groups.

Fig. 2

E4orf1 transiently but reproducibly enhances glucose excursion: C57BL/6J (9 week old) mice on 60% fat diet since 6 week of age were inoculated with pBabe-puro retrovirus (control; black solid lines, n = 6), or with pBabe expressing E4orf1 (black dotted lines; n = 6). Expression of E4orf1, enhanced blood glucose clearance 1 week later, compared to control group of mice (A) and the effect persisted for one more week (B). The effect of E4orf1 on GTT was transient and weeks 3 and 4 post-inoculation (p.i.) showed that the effect of first inoculation had faded (C–D). Upon re-inoculation in week7, GTT significantly improved again for the E4orf1 group 4 d (E) and a week later (F). (*p < 0.05, **p < 0.005).

Fig. 3

A booster dose of E4orf1 does not extend the duration of enhancement in glucose clearance: Nine week old C57BL/6J (9 wk old) male mice on 60% HF diet since 6 wk of age were inoculated with control retrovirus (black solid lines, n = 6), or with E4orf1 (black dotted lines; n = 6). GTT was performed 1 week p.i. (A–B) and mice were re-inoculated at 10 week of age and GTT performed 1 week post re-inoculation (C–D). The improved glucose clearance following each inoculation disappeared 3 week post initial inoculation (E–F) but a second booster inoculation again improved GTT for the E4orf1 group (G–H). (*p < 0.05, **p < 0.005). The bar graphs show area under the curve for respective groups.

Fig. 4

Longer duration of high fat diet delays the improvement in GTT induced by E4orf1: Fourteen week old C57BL/6J male mice on 60% fat diet (since 6wk) were inoculated with pBabe-puro (control; black solid lines/bars, n = 6), or with different doses of pBabe expressing E4orf1 (300 μL dose; black dotted lines/bar, n = 6 and 600 μL dose; black big dotted lines/bar, n = 6). E4orf1 expression does not improve blood glucose clearance 1 week p.i. (A–B). A booster dose of the respective vectors 3 week post initial inoculation significantly improved effect of E4orf1 on GTT (C–D). (*p < 0.05, **p < 0.005, ***p < 0.0005). The bar graphs show area under the curve for respective groups.

Fig. 5

E4orf1 improves glucose clearance without increasing insulin sensitivity, production or secretion: Insulin tolerance test (ITT) was performed 1 and 2 week post initial inoculation of control retrovirus (black solid lines, n = 3), or with E4orf1 (black dotted lines; n = 3) in 9 wk old HF fed C57Bl/6j male mice (A–D). The bar graphs (B & D) show area under the curve for respective groups. Serum insulin determined from fasted and non-fasted control (n = 3) and E4orf1 (n ¼ 3) inoculated mice (E). Groups were compared using ANOVA followed by tukey’s test (*p < 0.05). (F): 832/13 cells were transduced with the indicated viruses overnight. The 832/13 cells were incubated at the designated glucose concentrations for 2 h. Insulin secreted into the medium was measured by ELISA, normalized to total cellular protein, and was expressed as a fold increase relative to insulin secreted at 2 mmol/l glucose in cells that received no virus. Data represent the means ± SE for three independent experiments.

Fig. 6

E4orf1 enhances cell signaling in glucose disposal pathway: The glucose disposal pathway signaling in muscle, adipose tissue depots, and liver of control (n = 3) and E4orf (n = 3) mice was determined by western blotting. Muscle (A–B): p-Akt, p-AMPK, and Glut4 expressions were normalized to t-Akt, t-AMPK, and GAPDH, respectively. Adipose tissue (C–D): Glut4, adiponectin, RAS, PPARγ, expressions were normalized to GAPDH, p-Akt was normalized to t-Akt. Liver (E–F): Glut2, glucokinase expressions were normalized to GAPDH, p-Akt, p-AMPK were normalized to t-Akt or t-AMPK, respectively. Groups were compared using ANOVA followed by Tukey’s test. Only comparisons that were statistically significant (*p < 0.05, **p < 0.01) are presented as bar graphs.