Volume 15, Issue 1, Pages 86-93 (January 2007) Prolonged Remission of Diabetes by Regeneration of β Cells in Diabetic Mice Treated with Recombinant Adenoviral Vector Expressing Glucagon-like Peptide-1  Meng-Ju Liu, Seungjin Shin, Na Li, Toshikatsu Shigihara, Young-Sun Lee, Ji-Won Yoon, Hee-Sook Jun  Molecular Therapy  Volume 15, Issue 1, Pages 86-93 (January 2007) DOI: 10.1038/sj.mt.6300005 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Production of rAd-GLP-1. (a) The gene construct for a rAd-GLP-1. (b) The expression of exogenous GLP-1 mRNA in TTNT-16 cells was determined by RT-PCR 8 and 24 h after infection with rAd-GLP-1 or rAd-βGAL at MOI 10. The expression of GAPDH mRNA was analyzed as an internal control. Untreated TTNT-16 cells were used as a control. (c) Expression of GLP-1 in TTNT-16 cells was determined by immunohistochemical staining with GLP-1 antibody at 2 days after infection with 10 MOI of virus. (d) Secretion of GLP-1 into media was determined by RIA 60 h after infection of TTNT-16 cells with 10 or 100 MOI of virus. Values are mean±SD of three experiments. **P<0.0001. Molecular Therapy 2007 15, 86-93DOI: (10.1038/sj.mt.6300005) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 The expression of GLP-1 and insulin mRNA in various tissues in rAd-GLP-1-treated STZ-induced diabetic mice. STZ-induced diabetic NOD/SCID mice were injected with rAd-GLP-1 or rAd-βGAL, as a control. Various tissues were removed 2 weeks after viral injection, and GLP-1 and insulin mRNA were determined by RT-PCR. The expression of GAPDH mRNA was analyzed as an internal control. P: pancreas, L: liver, S: spleen, H: heart, Lu: lung. Representative data from three mice. Molecular Therapy 2007 15, 86-93DOI: (10.1038/sj.mt.6300005) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Remission of diabetes in rAd-GLP-1-treated STZ-induced diabetic mice. STZ-induced diabetic NOD/SCID mice (blood glucose levels>500 mg/dl) were injected with rAd-GLP-1 (STZ+rAd-GLP-1) or rAd-βGAL (STZ+rAd-βGAL), as a control. Untreated STZ-induced diabetic mice (STZ) and non-diabetic NOD/SCID mice served as controls. Normoglycemic rAd-GLP-1-treated mice were injected again with STZ at 30 days after virus injection. (a) Blood glucose levels were measured at various times after injection. (b) At 30 days after treatment, a glucose tolerance test was performed. Glucose (2 g/kg body weight, i.p.) was injected at 0 min and blood glucose levels were measured at the indicated times. Values are mean±SD. **P<0.0001 compared with STZ+rAd-βGAL and STZ groups. Molecular Therapy 2007 15, 86-93DOI: (10.1038/sj.mt.6300005) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Histological and immunohistochemical analysis of the pancreas in rAd-GLP-1-treated STZ-induced diabetic mice. STZ-induced diabetic NOD/SCID mice were treated with rAd-GLP-1 (STZ+rAd-GLP-1) or rAd-βGAL (STZ+rAd-βGAL). (a) 30 days after viral injection, serial pancreatic sections were stained with hematoxylin and eosin (HE) or double-stained with anti-insulin (red) and anti-glucagon (green) antibodies. (b) Mice were injected daily with BrdU (100 mg/kg body weight, i.p.) for 5 days beginning on the 8th day after virus injection. Pancreatic sections were prepared on the last day of BrdU injection and double-stained with anti-pdx-1 and anti-BrdU antibodies. Arrows indicate double-positive cells. Images were analyzed with a laser scanning confocal microscope. Representative islets are shown (original magnification × 400; bar=40 μm). (c) The insulin-positive area was measured after anti-insulin antibody staining at 30 days after virus injection and expressed as a percentage of the area found in normal mice. **P<0.001 compared with STZ+rAd-βGAL-treated group. Molecular Therapy 2007 15, 86-93DOI: (10.1038/sj.mt.6300005) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Serum insulin levels in rAd-GLP-1-treated STZ-induced diabetic mice. STZ-induced diabetic NOD/SCID mice treated with rAd-GLP-1 (STZ+GLP-1) or rAd-βGAL (STZ+rAd-βGAL). At 2 weeks after virus injection, mice were injected with glucose (2 g/kg body weight, i.p.), blood was collected at 30 min after glucose injection, and serum insulin concentrations were measured by EIA. Untreated STZ-induced diabetic mice (STZ) and non-diabetic NOD/SCID mice were used as controls (n=3/group). **P<0.0001 compared with STZ+rAd-βGAL and STZ groups. Molecular Therapy 2007 15, 86-93DOI: (10.1038/sj.mt.6300005) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 Remission of diabetes in autoimmune diabetic NOD mice by treatment with rAd-GLP-1 and an immunoregulator. (a) Autoimmune diabetic NOD mice (blood glucose levels>500 mg/dl) were injected with CFA (200 μl, subcutaneous). Three days later, rAd-GLP-1 (CFA+rAd-GLP-1) or rAd-βGAL (CFA+rAd-βGAL) (1 × 1012 viral particles) was injected, and blood glucose levels were measured. As a control, the mice were injected with CFA only (CFA) or rAd-GLP-1 only without CFA treatment (rAd-GLP-1). **P<0.0001 compared with CFA+rAd-βGAL-treated group. (b) Blood glucose levels were measured for 1 year in mice that achieved normoglycemia after treatment with CFA+rAd-GLP-1. (c) At 30 days after virus treatment, glucose tolerance tests were performed. Diabetic NOD mice and non-diabetic NOD mice (5-weeks-old) served as controls. **P<0.0001 compared with CFA+rAd-βGAL-treated group. Molecular Therapy 2007 15, 86-93DOI: (10.1038/sj.mt.6300005) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 7 Immunohistochemical analysis of pancreas in CFA and rAd-GLP-1-treated diabetic NOD mice. Pancreata were removed from autoimmune diabetic NOD mice at 30 days after treatment with CFA+rAd-GLP-1 or CFA+rAd-βGAL. (a) Adjacent sections were stained with hematoxylin and eosin (HE) or anti-insulin antibody (red). Inset shows the entire islet from a diabetic NOD mouse. Pancreatic sections from untreated diabetic NOD mice were used as a control. Original magnification × 400. (b) Pancreatic sections were stained with anti-C-peptide antibody. Pancreatic sections from non-diabetic mice were used as a control. Molecular Therapy 2007 15, 86-93DOI: (10.1038/sj.mt.6300005) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 8 Serum C-peptide levels in CFA and rAd-GLP-1-treated diabetic NOD mice. Diabetic NOD mice treated with CFA+rAd-GLP-1 or CFA+rAd-βGAL were injected with glucose, blood was collected at 30 min later, and serum C-peptide concentrations were measured. Non-diabetic normal mice were used as a control (n=3/group). **P<0.02 as compared with CFA+rAd-βGAL-treated group. Molecular Therapy 2007 15, 86-93DOI: (10.1038/sj.mt.6300005) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions