1. Volume 16, Issue 11, Pages 1805-1812 (November 2008)
Gene Therapy for Diabetes: Metabolic Effects of Helper-dependent Adenoviral Exendin 4 Expression in a Diet-induced Obesity Mouse Model 
Susan L Samson, Erica V Gonzalez, Vijay Yechoor, Mandeep Bajaj, Kazuhiro Oka, Lawrence Chan 
Molecular Therapy 
Volume 16, Issue 11, Pages (November 2008)
DOI: /mt
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
The exendin 4 (Ex4) cassette expresses immunoreactive and biologically active secreted Ex4. (a) Schematic diagram of the Ex4 expression cassette. (b) Transduction of Hep3B cells with HDAd-Ex4 (100 viral particles/cell) leads to detection of Ex4 in the cell lysate and the media after 48 hours as measured by enzyme immunoassay. (c) Rat 832/13 insulinoma cells were plated in 48-well dishes and cotransfected with 10 ng of pcDNA3.1 or pcDNA3.1-Ex4 (CMV-Ex4) and 400 ng of cyclic adenosine monophosphate–responsive element–driven luciferase reporter plasmid. After 48 hours, Ex4 peptide (10 nmol/l) or forskolin (10 µmol/l) was added in the indicated wells for an additional 6 hours before harvesting (*P < 0.05 compared to control). (d) 832/13 cells were transfected in quadruplicate with 10 ng of pcDNA3.1 or pcDNA3.1-Ex4. After 48 hours of expression, the media was removed and cells were treated with Hanks' Balanced Salt Solution (HBSS) containing low (2 mmol/l) or high (15 mmol/l) glucose for 4 hours, with or without Ex4 peptide (10 nmol/l) as indicated. Immunoreactive insulin secreted into the HBSS was measured by enzyme-linked immunosorbent assay and normalized to total cell protein for each well (*P < 0.05 indicated by brackets). LUC, luciferase.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
HDAd-Ex4 decreases weight gain and fat mass in diet-induced obesity (DIO) mice without detectable changes in food intake. The % weight change from baseline is shown with time from helper-dependent adenovirus (HDAd) injection (14 weeks of age) is shown for (a) DIO (n = 10 in each group) and (b) chow-fed mice (n = 5 in each group). The change was significant for the DIO mice (P < 0.05) at all time points. The net change in (c) fat mass and the (d) lean mass was measured by magnetic resonance imaging (*P < 0.05 HDAd-Ex4 compared to HDAd-0). (e) Twenty-four-hour food intakes were measured for 5 days at baseline (DIO, n = 20; chow, n = 10) and at 2 and 14 weeks after HDAd injection (DIO groups, n = 10; chow groups, n = 5) and are shown as kilocalories per gram of mouse per 24 hours. (f) Acute food intake was also measured after a 24-hours fast (n = 5 each group). Values are mean ± SD. Ex4, exendin 4.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
HDAd-Ex4 treatment alters metabolic rate. Indirect calorimetry was measured for singly housed mice 4 weeks post-treatment for 22 hours from 3 PM to 1 PM the next day. The dark cycle was from 8 PM to 6 AM. (a) VO2 (ml/kg/min) of diet-induced obesity (DIO) mice over the light and dark cycle (n = 7 each group). (b) Average VO2 (ml/kg/min) of chow and DIO mice (c) VO2 (ml/kg/min) of chow-fed mice over the light and dark cycle (n = 4 each group). (d) Calculated energy expenditure (EE). (e) The respiratory quotient (RQ) was calculated from the VCO2-to-VO2 ratio. The average VO2 and calculated energy expenditure (kJ/kg/min) is compared among the different treatment groups for the total 22-hours light and dark cycle as well as the 10-hour dark cycle alone (*P < 0.05 HDAd-Ex4 compared to HDAd-0). (f) Activity measurements. Values indicate mean ± SD. Ex4, exendin 4; HDAd, helper-dependent adenovirus.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Glucose homeostasis is improved by HDAd-Ex4. Intraperitoneal (IP) glucose tolerance tests were performed (1.5 mg glucose/g mouse IP) for (a) diet-induced obesity (DIO) mice (n = 10, each group) and (b) chow-fed mice (n = 5, each group) at 15 weeks post-treatment. The area under the curve (AUC) for (c) blood glucose and (d) plasma insulin was calculated (*P < 0.05 HDAd-Ex4 compared to helper-dependent adenovirus (HDAd)-0 at the same time point). Acute insulin secretion (3.0 mg glucose/g mouse IP) was performed for at 15 weeks post-HDAd injection for DIO HDAd-treated mice (n = 10, each group), chow-fed mice (n = 5) and DIO mice injected with 2.4 nmol/kg mouse of commercial exendin 4 (Ex4) (n = 5) for measurement of (e) blood glucose and (f) plasma insulin levels (*P < 0.05 HDAd-0 compared to HDAd-Ex4; **P <0.05 for Ex4 peptide injection compared to both HDAd-Ex4 and HDAd-0). (g) The product of the fasting insulin × fasting glucose was calculated (*P < 0.05). (h) An insulin tolerance test was performed with 1.0 unit/kg mouse insulin IP. (i) Relative liver RNA levels of glucose-6-phosphatase (G6P) and PEPCK were measured by quantitative PCR and normalized to cyclophilin. The HDAd-0 values were set at 1.0 for comparison with HDAd-Ex4 (*P < 0.05). Values are mean ± SD.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Adipokine levels are altered by HDAd-Ex4 treatment. (a) Fasting (24 hours) plasma levels of adiponectin (Adpn; µg/ml), leptin (ng/ml), and resistin (ng/ml) are shown at 10 weeks after helper-dependent adenovirus (HDAd) injection for diet-induced obesity (DIO) mice (n = 10 each group; *P < 0.05 for HDAd-Ex4 compared to HDAd-0). Values are mean ±SD. Scatter plots of (b) resistin versus % fat and (c) adiponectin versus % fat are shown (HDAd-0, white circles; HDAd-Ex4, black circles). Ex4, exendin 4.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Hepatic fat is decreased in HDAd-Ex4-treated mice. (a) Liver tissue from mice treated for 15 weeks with helper-dependent adenovirus (HDAd)-0 or HDAd-Ex4 were fixed in phosphate-buffered formalin, paraffin sectioned, and hematoxylin and eosin staining was performed. Mice were fasted for 4 hours before killing. Representative sections from three separate mice for each HDAd treatment group are shown. Bars = 50 µm. (b) Lipid and triglyceride content of the liver was measured (n = 5/group; *P < 0.05 compared to baseline and brackets indicate P < 0.05 HDAd-Ex4 compared to HDAd-0). (c) The relative liver RNA expression of levels of key lipogenic gene products were measured by quantitative PCR normalized to cyclophilin (*P < 0.05 for HDAd-Ex4 compared to HDAd-0). Mice were fasted for 4 hours before killing. Values are mean SD. Ex4, exendin 4. Fasn, fatty acid synthase; Scd1, stearoyl coA desaturase 1; Acc, acetyl CoA carboxylase; Dgat, diacylglycerol acetyltransferase; Mgat, monoacylglycerol acetyltransferase; Fabp1, Fatty acid–binding protein 1; PPAR, peroxisome proliferator–activated receptor; SREBP-1c, sterol regulatory-element-binding protein-1c.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions