Gene Transfer of Constitutively Active Akt Markedly Improves Human Islet Transplant Outcomes in Diabetic Severe Combined Immunodeficient Mice

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FIG. 1.FIG. 1.
FIG. 1.

Akt expression, phosphorylation, and activity in human islets after transduction with Adv-CA-Akt. Effect of different doses of the adenovirus on insulin secretion. A: Expression of CA-Akt was assessed by immunofluorescence in sections of human islets transduced with Adv-CA-Akt or Adv-LacZ stained with antibodies against the hemagglutinin epitope Tag (green) and insulin (red). B: Expression and phosphorylation of Akt was assessed by Western blot analysis of protein extracts obtained from uninfected human islets and human islets transduced with different MOI of Adv-LacZ or Adv-CA-Akt. Twenty-four hours after transduction, islets were harvested, and protein was extracted. To detect Akt phosphorylation, antibodies against phosphorylated Ser473 and Thr308 were used. Actin was used as an internal control for loading. C: Akt activity was measured in human islets transduced with 500 MOI of Adv-LacZ or Adv-CA-Akt and in uninfected islets. Twenty-four hours after transduction, islets were harvested, and protein was extracted. The level of GSK-3α/β phosphorylation was used to measure Akt activity as described under research design and methods. A representative immnuoblot of five independent experiments using phospho-GSK-3α/β (Ser 21/9) antibody is shown. D: Glucose-stimulated insulin secretion in uninfected and adenovirus-transduced islets. Human islets were exposed to 500 or 1,000 MOI of Adv-LacZ and Adv-CA-Akt for 1 h. Twenty-four hours after transduction, groups of 10 islets of similar sizes were incubated with 5 or 22 mmol/l glucose for 30 min, and insulin secreted in the media was measured by radioimmunoassay. Results are the means ± SE of six to eight different experiments performed in triplicate. The data are presented as percentage above the insulin secreted by uninfected islets (100%). *P < 0.05 vs. the corresponding insulin secretion at 5 mmol/l glucose.

This Article

  1. Diabetes vol. 54 no. 6 1664-1675