Efficient and controlled gene expression in mouse pancreatic islets by arterial delivery of tetracycline-inducible adenoviral vectors

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  • 1 Division of Molecular Metabolism and Diabetes, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
  • 2 1Advanced Therapeutics for Metabolic Diseases, Center for Translational and Advanced Animal Research, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan

Gene transfer with adenovirus vectors has been used extensively for pancreatic islet research. However, infection efficiency varies among reports. We reevaluated the infection efficiency, defined here as the percentage of islet cells expressing transgenes, in mouse islets. When the isolated islets were infected with adenoviruses, the infection efficiency was found to be 30–40% and the transduced cells were distributed in the islet periphery. Collagenase treatment of isolated islets before infection increased the infection efficiency to 70%, but with suppression of glucose-stimulated insulin secretion. To explore more efficient strategies, we employed arterial delivery of virus particles to islets in situ. Delivery of adenovirus (~108 particles per pancreas) through the celiac and superior mesenteric arteries is highly efficient, resulting in more than 90% transduction without impairing glucose-stimulated insulin secretion. Arterial delivery of an adenovirus harboring glycerol kinase cDNA allowed us to observe glycerol-stimulated insulin secretion from mouse islets, which was not observed when we employed the conventional method. Furthermore, the arterial delivery method combined with a tetracycline-inducible adenovirus system induced efficient and controlled transgene expression. Our data provide new insights into gene transduction methods using recombinant adenoviruses in mouse islets, and are therefore anticipated to contribute to future basic and clinical islet research applications.

Abstract

Gene transfer with adenovirus vectors has been used extensively for pancreatic islet research. However, infection efficiency varies among reports. We reevaluated the infection efficiency, defined here as the percentage of islet cells expressing transgenes, in mouse islets. When the isolated islets were infected with adenoviruses, the infection efficiency was found to be 30–40% and the transduced cells were distributed in the islet periphery. Collagenase treatment of isolated islets before infection increased the infection efficiency to 70%, but with suppression of glucose-stimulated insulin secretion. To explore more efficient strategies, we employed arterial delivery of virus particles to islets in situ. Delivery of adenovirus (~108 particles per pancreas) through the celiac and superior mesenteric arteries is highly efficient, resulting in more than 90% transduction without impairing glucose-stimulated insulin secretion. Arterial delivery of an adenovirus harboring glycerol kinase cDNA allowed us to observe glycerol-stimulated insulin secretion from mouse islets, which was not observed when we employed the conventional method. Furthermore, the arterial delivery method combined with a tetracycline-inducible adenovirus system induced efficient and controlled transgene expression. Our data provide new insights into gene transduction methods using recombinant adenoviruses in mouse islets, and are therefore anticipated to contribute to future basic and clinical islet research applications.

Introduction

Gene therapy holds promise for the treatment of many human diseases, including diabetes mellitus (Yechoor & Chan 2005). Ex vivo gene transfer to pancreatic β-cells offers a potential means of preventing β-cell death via the expression of immunoregulatory, cytoprotective, or anti-apoptotic genes (Fernandes et al. 2004, Tran et al. 2004). In addition, the induction of transcriptional regulators and growth factors by gene therapy could stimulate β-cell differentiation and regeneration (Brun et al. 2004, Cozar-Castellano et al. 2004). Among several gene transfer vehicles, adenovirus vectors are attractive because they have the capacity for large DNA inserts, can be produced at high titers and have abilities to transduce non-dividing cells. In addition to potential therapeutic uses, adenovirus vectors are valuable experimental tools for genetic manipulation of islet cells. However, using adenovirus vectors, infection efficiency, defined here as the percentage of cells expressing transgenes, are reportedly variable, ranging from 10 to 80% in human (Giannoukakis et al. 1999, Leibowitz et al. 1999, Barbu et al. 2005, Rao et al. 2005), rat (Becker et al. 1994, Weber et al. 1997, Ishihara et al. 2003, Zhou et al. 2003, Diraison et al. 2004), and mouse (Csete et al. 1995, Bertera et al. 2003, Garcia-Ocana et al. 2003, Leclerc et al. 2004, Diao et al. 2005) islets.

There exist large numbers of genetically modified mice and basic aspects of mouse pancreatic islets have been intensively studied. Therefore, mouse islets are a suitable model for applying adenovirus technology, with the goal of clinical application of ex vivo gene transfer for this endocrine organ. For certain purposes, including transfection of cytoprotective genes for islet transplantation, it is essential that most of the cells express the transgenes. High efficiency is also desirable for studying islet biology and essential for suppressing gene expression in islets with adenovirus-mediated shRNA expression (Bain et al. 2004, Diao et al. 2005). In this study, we first reevaluated the transfection efficiency of recombinant adenoviruses in mouse islets and then explored the usefulness of different strategies, collagenase treatment before infection, and arterial delivery of virus particles in situ. We observed markedly improved infection efficiency with these methods. In particular, arterial delivery combined with a tetracycline-inducible adenovirus system induced reliable and controlled expression of foreign genes in mouse islets.

Material and methods

Mouse

C57BL/6 mice, 12–16 weeks of age, were used. All animal experiments were approved by the Tohoku University Institutional Animal Care and Use Committee (#15–110).

Recombinant adenoviruses

AdRIP-HArGlyK expressing hemagglutinin (HA) epitope-tagged rat glycerol kinase under control of the rat insulin 1 promoter was described previously (Takahashi et al. 2006). The cytomegalovirus (CMV) promoter containing the Tet operator sequence (CTO) and the enhanced green fluorescent protein (eGFP) cDNA were excised from pcDNA5/TO (Invitrogen) and pEGFP-1 (BD Biosciences Clontech) respectively, and then ligated. The Tet-repressor cDNA was excised from pcDNA6/TR (Invitrogen) and ligated to the CAG promoter unit (Niwa et al. 1991). The CMV-eGFP expression unit was excised from pEGFP-1. These expression units were cloned into a cosmid vector pAdex1cw (Miyake et al. 1996). These cosmid vectors containing the expression units and adenovirus DNA-terminal protein complex were then co-transfected into HEK293 cells, which were then seeded onto 96-well plates. After 10 days, cytopathic effects were seen in several wells. Recombinant adenoviruses were extracted from 96-well plates and amplified first in HEK293 cells in 24-well plates and then in cells in 3×150 mm dishes. Usually, we pick up adenoviruses from 96-well plates in which no more than 20 wells show cytopathic effects. In addition, careful examination of adenovirus DNA structure by enzyme digestion excludes contamination with adenoviruses lacking some portion of their genome. Therefore, we consider the purity ofthe virus preparation tobe similarto that obtained from purified plaques. Amplified adenoviruses were purified with CsCl gradient ultracentrifugation. The resulting viruses were designated AdCAG-TR for the Tet repressor expressing virus, AdCMV-eGFP for the eGFP-expressing virus under the CMV promoter, and AdCTO-eGFP for the eGFP-expressing virus under the CMV promoter with the Tet operator. Virus infectious titers (plaque-forming unit (pfu)) were determined by a previously described method (Miyake et al. 1996). Amounts of virus particles were calculated from OD260 with a formula in which 1 OD260 corresponds to 1.1×1012 particles/ml (Maizel et al. 1968). Viral particle to pfu ratios of these virus stocks were 20–80. Infectious titer (pfu) is more closely related to viral infectivity and thus used to present the virus amount in this study.

Isolation of mouse islets and infection with recombinant adenoviruses

Islets were isolated by retrograde infusion of 1.3 ml cold Hanks’-balanced salt solution containing 1.0 mg/ml collagenase (Sigma-Aldrich) from the common bile duct and harvested by hand under microscopy. One hundred islets, immediately after isolation or after overnight culture, were placed in 1 ml RPMI media containing 10% fetal bovine serum, 11 mM glucose, 100 U/ml penicillin, and 100 μg/ml streptomycin, to which recombinant adenoviruses were added at an m.o.i. of 300 (assuming 2500 cells per islet; 7.5×107 pfu of viruses/100 islets per milliliter), and cultured at 37 °C for 2 h. Islets were then washed twice with the media and cultured for 48 h in RPMI media. In some of the experiments, islets cultured overnight were treated with 1 ml Hanks’-balanced salt solution containing 1.0 mg/ml collagenase for 10 min at 37 °C, washed twice with PBS and then infected with recombinant adenoviruses as mentioned earlier.

Arterial delivery of adenovirus vectors

For arterial delivery of virus particles, polyethylene tubes 0.3 mm in diameter were inserted into the celiac artery (CA) and the superior mesenteric artery (SMA) (Fig. 1). Viruses (0.5 to 5×108 pfu) were diluted in 1.3 ml saline. After clamping the portal vein and the hepatic artery as well as the splenic artery, an approximately 0.9 ml viral solution was injected into the pancreas via the CA and 0.4 ml through the SMA. The pancreas was left at room temperature for 10 min and then 1.3 ml cold Hanks’-balanced salt solution containing 1.0 mg/ml collagenase was infused through the common bile duct. Islets were harvested by hand and cultured for 48 h.

GFP expression and immunocytochemistry of islet sections

Islets were fixed with 4% paraformaldehyde for 15 min at 4 °C, immersed in 5% sucrose/PBS for 30 min and 30% sucrose/PBS for 1 h. Samples were then embedded in TISSU MOUNT (Chiba Medical, Saitama, Japan) and quick-frozen in liquid nitrogen. Islet sections (6 μm) were made using a cryostat and examined for eGFP fluorescence with a Leica fluorescent microscope (DFC350FX). Sections were also stained with anti-insulin and anti-glucagon antibodies (Sigma-Aldrich).

Flow cytometric analysis of GFP expression

The infected islet cells were quantitatively analyzed by flow cytometry (FACS Calibur; BD Bioscience Clontech) on at least 1×104 cells per sample. Forty-eight hours after infection, islet cells were dispersed by treatment with 0.25% trypsin per 1 mM EDTA for 5 min at 37 °C. The percentage of eGFP-positive cells (in the M2 range) was determined after compensating for autofluorescence (in the M1 range) using uninfected cells as a negative control.

Measurement of insulin secretion

Islets (ten islets per tube) infected with recombinant adenoviruses were incubated over a period of 60 min in 1 ml Krebs–Ringer bicarbonate Hepes buffer (KRBH, 140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 2 mM NaHCO3, 10 mM Hepes (pH 7.4), 0.25% BSA) containing 2.5 mM glucose, 2.5 mM glucose plus 10 mM glycerol, 15 mM glucose or 20 mM glucose. Experiments were conducted with three to five tubes for each condition. Insulin was detected using RIA kits (LINCO, St Charles, MO, USA).

Statistical analysis

Data are presented as mean ± s.e.m. when otherwise stated. Differences between groups were assessed by Student’s t-test for paired or unpaired data.

Results

Adenovirus-mediated gene transfer using the conventional method

We reevaluated the efficiency of adenovirus infection in isolated mouse islets. An optimal titer of adenoviruses was first determined employing AdCMVeGFP expressing the enhanced Green fluorescent protein (eGFP). Analysis using a fluorescence-activated cell sorter (FACS) showed the percentage of islet cells expressing eGFP to be increased, as virus titer rose from multiplicity of infection (m.o.i.) of 100–900 (Fig. 2A). In addition, the expression levels, as evaluated by geometric mean fluorescence, were increased from 46.46 ± 0.54 (m.o.i.=100), 71.98 ± 5.79 (m.o.i.=300) to 111.26 ± 6.56 (m.o.i.=900; arbitrary unit n=3). However, the infection of islets with AdCMV-eGFP at an m.o.i. of 900 resulted in reduced glucose (20 mM)-stimulated insulin secretion when compared with that in non-infected islets (data not shown), as was noted previously (Rao et al. 2005). In contrast, a more than fourfold increase in glucose-stimulated insulin secretion was observed in both non-infected islets and those infected with AdCMV-eGFP at an m.o.i. of 100 or 300 (data not shown). Therefore, for the subsequent studies, we used AdCMV-eGFP at an m.o.i. of 300.

Many other parameters affect infection efficiency, such as islet size and duration of exposure to the adenovirus. As described previously (Leclerc et al. 2004), we noticed that cells at the centers of islets with a diameter >150 μm were difficult to infect. Therefore, islets with a diameter between approximately 80 and 120 μm were employed for the present study. Very little necrosis of cells at the islet core (central necrosis) was seen in islets with this size range (data not shown). As to the duration of exposure to the adenovirus, we observed that 2-h exposure to the virus at 37 °C resulted in greater numbers of infected cells than 1-h exposure, while no further increases were seen with overnight exposure (data not shown).

Another factor that could affect the infection efficiency is the timing of adenovirus infection. Therefore, we examined whether overnight culture affects the infection efficiency in mouse islets. Islets were infected with AdCMV-eGFP either immediately after isolation or after an overnight culture. Some eGFP-positive cells were observed in the inner parts of islets when islets were infected immediately after isolation (Fig. 2B–D), while eGFP-positive cells were located almost exclusively in the surface layer of islets when infected after an overnight culture (Fig. 2E–G). Quantitative analysis using a FACS demonstrated that eGFP-positive cell numbers tended to be greater in islets infected immediately after isolation (39.8 ± 2.4%, n=3) than in those infected after an overnight culture (35.6 ± 1.2%, n=3), but the difference did not reach statistical significance. Immunostaining with an insulin or glucagon antibody revealed that small percentages of β-cells were infected either immediately after isolation (Fig. 2C) or after an overnight culture (Fig. 2F), while very high percentages of α-cells, nearly 100%, were infected. The latter observation was especially apparent in islets infected immediately after isolation (Fig. 2D and G).

Islets were fragmented or damaged with a few cells detaching from the islet surface immediately after isolation (data not shown). In contrast, islets appeared to have recovered, forming compact spheres, after an overnight culture. We speculated that the somewhat higher infection efficiency in islets immediately after isolation might be related to islet surface damage, which allows virus entry into the inner parts of islets.

Increased efficiency of adenovirus infection after collagenase treatment

To improve the infection efficiency, islets cultured overnight were treated with 1.0 mg/ml collagenase for 10 min at 37 °C before adenovirus infection. Infected islets are then cultured for 48 h. We observed eGFP-positive cells even at the islet center (Fig. 3A–C) and recognized a marked increase in the percentage of eGFP-positive cells; average infection efficiency was 73.24 ± 5.30% (n=3). Representative data of the FACS analysis were shown in Fig. 3D. Consequently, not only α-cells, but also the majority of β-cells expressed eGFP (Fig. 3B and C). We also tested 0.2 and 0.5 mg/ml collagenase, but found efficiency to be lower than with 1.0 mg/ml collagenase (data not shown).

We first confirmed that insulin secretion was not affected by treatment with 1.0 μg/ml collagenase (Fig. 3E). We then studied the effects of adenovirus infection after collagenase treatment on glucose-stimulated insulin secretion. Glucose responsiveness was preserved as judged by similar ratios of stimulated over basal insulin secretion in control islets (5.27 ± 0.50-fold, n=3) and islets infected with AdCMV-eGFP after collagenase treatment (5.29 ± 0.81-fold, n=3; Fig. 3F). However, absolute amounts of insulin secreted from islets were significantly lower when islets were infected after collagenase treatment than those of non-infected and non-treated islets (Fig. 3F, open columns versus closed columns). The decrease in insulin secretion was not attributable to reduced insulin contents, since insulin contents did not differ between these islets (226.3 ± 46.5 vs 228.5 ± 35.4 ng/islet, n=3 experiments, for control and infected islets respectively). Considering that the fold-increase in glucose-stimulated insulin secretion and insulin content in infected islets were similar to those in non-infected islets, glucose-sensing, and the associated intracellular signaling were thought to be unaffected, while insulin exocytosis was impaired by adenovirus infection after collagenase treatment.

Arterial delivery of recombinant adenovirus increased gene transduction efficiency

Next, to devise a better infection method, we examined gene transduction efficiency with adenovirus delivery to mouse islets in situ through arteries perfusing the pancreas. Arterial delivery of recombinant adenoviruses has been used to infect islets of obese Zucker diabetic fatty rats (Wang et al. 1998). However, neither the details of the method used nor its infection efficiency have been described. Furthermore, arterial delivery has not been applied for mouse islet infection. Therefore, we tested the feasibility of delivering recombinant adenoviruses through both the celiac artery (CA) and the superior mesenteric artery (SMA). The SMA perfuses mainly the ventral portion of the pancreas, while the CA supplies the pancreatic body and tail. With the portal vein and the hepatic artery as well as the splenic artery clamped, 3.4×108 plaque forming units (pfu) of AdCMV-eGFP virus were delivered through these arteries: 70% through the CA and 30% through the SMA (Fig. 1). The pancreas was then left at room temperature for 10 min. The islets were next isolated by retrograde collagenase infusion through the common bile duct. More than 90% of islets showed viral infection with this method emitting green fluorescence (Fig. 4A). Importantly, eGFP-positive cells were distributed throughout almost all infected islets, and the intensity of green fluorescence was homogenous (Fig. 4A–C). Furthermore, both β-cells (Fig. 4B) and α-cells (Fig. 4C) were effectively infected. Quantitative assessment of efficiency, determined using FACS, revealed 90.37 ± 2.0% (n=3) of islet cells to be infected with the adenovirus. The expression levels of eGFP in individual cells were very high as shown by the rightward shift of the peak fluorescence in the FACS analysis (Fig. 4D compared with Fig. 3D). In addition, glucose-stimulated insulin secretion was unaffected by infection with adenovirus delivered through the CA and the SMA, being essentially the same as that in non-infected control islets (Fig. 4E).

We then studied the impact of rat glycerol kinase expression (rGlyK), by the arterial delivery method, on β-cell-secretory functions. We previously reported that glycerol stimulates insulin secretion from INS-1E cells and rat β-cells overexpressing rGlyK (Takahashi et al. 2006). However, when mouse islets were infected immediately after isolation with AdRIP-HArGlyK expressing rGlyK (Fig. 5A), we detected no glycerol-stimulated insulin secretion (Fig. 5B). We considered this to be due to low efficiency of adenovirus infection into mouse β-cells with the conventional method (Fig. 5A). Therefore, mouse islets were infected with AdRIP-HArGlyK via delivery through the CA and the SMA. When islets were infected with arterially delivered viruses, almost all β-cells expressed HA-GlyK (Fig. 5C). As expected, glycerol (10 mM) evoked insulin secretion from mouse islets infected with AdRIP-HArGlyK delivered through these arteries (Fig. 5D). These data, taken together, indicate that adenovirus administration through the CA and the SMA allows very high infection efficiency without adverse effects on islet-secretory activities.

Controlled gene expression with tetracycline-inducible adenoviruses

In comparing the effects of different adenovirus transductions, it is essential to use fixed amounts of adenoviruses, which do not differ between control and experimental groups. However, this is not an easy task. To create comparable conditions when using the arterial delivery method, a tetracycline-inducible system was applied. This system employs an adenovirus expressing the Tet repressor (AdCAG-TR) and another virus with a promoter containing the Tet operator. AdCTO-eGFP (1.5×108 pfu) expressing eGFP under control of the CMV promoter containing the Tet operator was delivered together with AdCAG-TR (0.5×108 pfu) into the pancreas through the CA and the SMA. Islets were then isolated, divided into two groups and incubated in media with or without doxycycline (2 μg/ml). Islets treated with doxycycline exhibited strong eGFP expression, while only weak expression in a limited number of cells was observed in islets infected with these viruses but not treated with doxycycline (Fig. 6). These data indicate that the arterial delivery method combined with the tetracycline-inducible system assure efficient and controlled gene expression in islets.

Discussion

We studied the effects of different infection methods on adenovirus infection efficiency in mouse islets, including virus delivery through the CA and the SMA. Several factors influence the efficiency of adenovirus infection and cause variable levels of cytotoxicity. Results should always be interpreted with caution, and limited infection efficiency and possible cytotoxicity must be taken into account.

The efficiency of adenovirus infection in pancreatic islets varies among reports. Since not all factors in previous studies were provided, reasons for the different efficiencies are unclear. In this study, we showed that collagenase treatment of islets before exposure to viruses improves the efficiency. High efficiencies in several studies (Becker et al. 1994, Giannoukakis et al. 1999, Ishihara et al. 2003, Zhou et al. 2003, Diao et al. 2005) could be due to surface damage by collagenase treatment during isolation. Necrosis of cells at the islet core (central necrosis) is sometimes observed and could affect infection efficiency (Ilieva et al. 1999, Giuliani et al. 2005). When efficiency is estimated by counting dispersed islet cells expressing transgenes, central necrosis could result in an erroneous increase in efficiency. This is because there is a possibility that uninfected necrotic cells located in the islet core were lost during dispersion. Central necrosis occurs more often in relatively larger islets (Ilieva et al. 1999). In this respect, rat or human islets appear to be more susceptible to central necrosis than mouse islets. In mice, as demonstrated in this study using islets 80–120 μm in diameter, central necrosis is not a major problem.

Several methods have been employed to enhance adenoviral transgene expression. Adenoviruses enter cells via the coxsackievirus and adenovirus receptor (CAR). Use of a modified adenovirus that has an Arg-Gly-Asp (RGD) motif in the adenovirus fiber knob markedly improves infection efficiency in nonhuman primate isolated islets (Bilbao et al. 2002). We infected mouse islets with an eGFP-expressing adenovirus containing the RGD motif but observed no improvement in efficiency (R Takahashi, H Ishihara and H Mizuguchi, unpublished observation). Very high infection efficiency in monolayer culture of mouse islet cells suggests sufficient expression levels of CAR in mouse islet cells (Narushima et al. 2004). In addition, several agents were reported to enhance transgene expression in other cell types ( Jornot et al. 2002, Huang et al. 2005, Triplett et al. 2005), and these agents may also be useful for pancreatic islet cells.

Arterial delivery of adenovirus was demonstrated to be highly efficient, although the procedure is not as easy as infection of isolated islets in vitro. Since nearly 100% infection is not possible with conventional infection methods, this method could be useful when nearly 100% infection is desirable, such as gene silencing by expression of short hairpin RNA using adenoviral vectors. It is also likely that this method can be more easily applied to larger animals, including humans. In the present study, more than 90% infection was achieved by arterial delivery of approximately 3×108 (plaque forming units (pfu)) viruses. This is much less than the amount used in Zucker diabetic fatty rats (1×1012 pfu; Wang et al. 1998), even after taking into account the differences in animal sizes. The mouse pancreas contains more than 1000 islets, including small islets. On the assumption that average cell number in each islet is 1000 (not 2500 because of the presence of small islets), the administration of 3×108 viruses into a pancreas corresponds to an m.o.i. of 300. In fact, the m.o.i. in our experiments must have been <300, since the virus solution was distributed not only to islets but also to acinar cells. This might explain the lack of adverse effects on insulin secretion in association with virus infection by this method. Although the arterial delivery method is promising and the conditions we used in this study are acceptable in terms of the transfection efficiency, functional impact, and lack of adverse effects, further studies are needed to optimize conditions. In this regard, we found that the delivery of viruses at more than 3×109 pfu resulted in blunted glucose-stimulated insulin secretion and that more than 3 ml adenovirus solution cannot be infused when portal vein, hepatic artery, and splenic artery are clamped.

We also demonstrated combining arterial delivery of the virus with a tetracycline-inducible expression system to enhance the feasibility of the arterial delivery method. This method allows us to use exactly the same amount of adenovirus in the control and experimental groups, avoiding false results due to possible differences in titers of control and experimental viruses. As discussed in an earlier report (Irminger et al. 1996), there is inherent inaccuracy in determining adenovirus titers. The inducible adenovirus vector system is also advantageous since phenotype changes can be analyzed according to the degree of gain or loss of gene expression.

In conclusion, the present data provide new insight into gene transduction using recombinant adenoviruses in mouse islets. Our data demonstrate that adenovirus administration through the CA and the SMA yielded very high transduction efficiency in mouse islets. This method could easily be applied to human pancreas removed from donors prior to isolating islets. Several genes have been demonstrated to improve islet transplantation performance (Bertera et al. 2003, Garcia-Ocana et al. 2003, Fernandes et al. 2004). Our data could thus contribute to future studies in β-cell research, and may well be applicable to islet transplantation.

Figure 1
Figure 1

Arterial delivery of recombinant adenovirus into mouse pancreas. Polyethylene tubes 0.3 mm in diameter were inserted into the celiac artery (CA, triangle) and the superior mesenteric artery (SMA, arrowhead) at the points branching from the aorta. The pancreas (P) was infused with virus solution and thereby distended. Sp, spleen; St, stomach; L, liver.

Citation: Journal of Molecular Endocrinology 38, 1; 10.1677/jme.1.02189

Figure 2
Figure 2

Adenovirus-mediated eGFP expression in mouse islets. (A) Mouse islets were infected with AdCMV-eGFP immediately after isolation at an m.o.i. of 100, 300, or 900. Percentages of eGFP-positive cells were analyzed by FACS 48 h after infection. Data are means ± s.d. of three independent experiments. (B–D) Immunostaining of sections of mouse islets infected with AdCMV-eGFP (300 m.o.i.) immediately after isolation. Sections of islets were directly observed for eGFP at low magnification (×100) (B) or stained with anti-insulin (C) or anti-glucagon (D) and observed at high magnification (×400). (E–G) Immunostaining of sections of mouse islets infected with AdCMV-eGFP (300 m.o.i.) after an overnight culture. Sections of islets were directly observed for eGFP at low magnification (×100) (E) or stained with anti-insulin (F) or anti-glucagon (G) and observed at high magnification (×400). Bars, 100 μm in B and E, and 20 μm in C, D, F and G.

Citation: Journal of Molecular Endocrinology 38, 1; 10.1677/jme.1.02189

Figure 3
Figure 3

Effects of collagenase treatment on AdCMV-eGFP infection in overnight-cultured islets. (A–C) Immunostaining of sections of mouse islets infected with AdCMV-eGFP after collagenase treatment. Sections of islets were directly observed for eGFP at low magnification (×100) (A) or stained with anti-insulin (B) or anti-glucagon (C) and observed at high magnification (×400). Bars, 100 μm in A and 20 μm in B and C. (D) Flow cytometric analysis of eGFP-positive cells in islets infected after collagenase treatment. One representative result from three experiments is presented. (E) Glucose-stimulated insulin secretion from collagenase-treated islets (closed columns) and non-treated control islets (open columns). The value of insulin secretion from control islets at 2.5 mM glucose (6.54 ± 0.72 ng/h per ten islets, n=3) was taken as 100%. Data are means ± s.e.m. from three independent experiments. (F) Glucose-stimulated insulin secretion from islets infected with AdCMV-eGFP after an overnight culture with collagenase treatment (closed columns) and that of control islets, which were neither infected nor treated with collagenase (open columns). The values of insulin secretion from control islets at 2.5 mM glucose (7.01 ± 0.64 ng/h per ten islets, n=3) was taken as 100%. Data are means ± s.e.m. from three independent experiments. *P<0.05, P<0.01.

Citation: Journal of Molecular Endocrinology 38, 1; 10.1677/jme.1.02189

Figure 4
Figure 4

Efficient gene transduction through arterial delivery of adenovirus vectors. (A–C) Immunostaining of sections of mouse islets infected with AdCMV-eGFP delivered through the CA and the SMA. Sections of islets were directly observed for eGFP at low magnification (×100) (A) or stained with anti-insulin (B) or anti-glucagon (C) and observed at high magnification (×400). Bars, 100 μm in A and 20 μm in B and C. (D) Flow cytometric analysis of eGFP-positive cells in islets infected with AdCMV-eGFP delivered through the CA and the SMA. One representative result out of three is shown. (E) Glucose-stimulated insulin secretion from islets infected with AdCMV-eGFP delivered through the CA and the SMA (closed columns) and that of non-infected islets (open columns). The value of insulin secretion from control islets at 2.5 mM glucose (2.89 ± 0.68 ng/h per ten islets, n=3) was taken as 100%. Data are means ± s.e.m. from three independent experiments.

Citation: Journal of Molecular Endocrinology 38, 1; 10.1677/jme.1.02189

Figure 5
Figure 5

Impact of efficient gene expression, by arterial delivery of recombinant adenovirus, on insulin secretion. (A) Islets infected with AdRIP-HArGlyK immediately after isolation were cultured for 48 h, dispersed and stained with anti-HA or anti-insulin. Experiments were repeated thrice with essentially similar results. (B) Glucose (Glc; 20 mM) or glycerol (Gly; 10 mM)-stimulated insulin secretion from islets infected with AdRIP-HArGlyK immediately after isolation (closed columns) and non-infected control islets (open columns). The value of insulin secretion from control islets at 2.5 mM glucose (6.30 ± 0.77 ng/h per ten islets, n=4) was taken as 100%. Data are means ± s.e.m. from four independent experiments. (C) Islets infected with AdRIP-HArGlyK, delivered through the CA and the SMA, were cultured for 48 h, dispersed and stained with anti-HA or anti-insulin. Experiments were repeated twice with essentially similar results. (D) Glucose (20 mM) or glycerol (10 mM)-stimulated insulin secretion from islets infected with arterially delivered AdRIP-HArGlyK (closed columns) and non-infected control islets (open columns). The value of insulin secretion from control islets at 2.5 mM glucose (3.51 ± 0.63 ng/h per ten islets, n=3) was taken as 100%. Data are means ± s.e.m. from three independent experiments. *P<0.05.

Citation: Journal of Molecular Endocrinology 38, 1; 10.1677/jme.1.02189

Figure 6
Figure 6

Doxycycline-inducible eGFP expression via adenoviruses delivered through the CA and the SMA. AdCAG-TR (0.5×108 pfu) and AdCTO-eGFP (1.5×108 pfu) were injected into the pancreas through the CA and the SMA. Expression of eGFP was induced by treatment with doxycycline (DOX, 2 μg/ml) for 48 h. The pictures shown are representative of three independent experiments. Bars, 20 μm.

Citation: Journal of Molecular Endocrinology 38, 1; 10.1677/jme.1.02189

We thank Prof. T Ito (Tohoku University) for his helpful advice on the morphological studies. Dr A Gjinovci (University of Geneva) is greatly acknowledged for his advice on perfusion methodology. We are also grateful to Y Nagura and K Tanaka for their expert technical assistance.

Funding

This study was supported by a Grant-in-Aid for Scientific Research (17590264 to H I) and the 21st Century COE Programs (‘the Center for Innovative Therapeutic Development for Common Diseases’) to Y O from the Ministry of Education, Science, Sports and Culture of Japan. This work was also supported by a Grant-in-Aid for Scientific Research (H16-genome-003) to Y O from the Ministry of Health, Labor and Welfare of Japan. There is no conflict of interest that would prejudice the impartiality of any of the authors of this manuscript.

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  • Brun T, Franklin I, St-Onge L, Biason-Lauber A, Schoenle EJ, Wollheim CB & Gauthier BR 2004 The diabetes-linked transcription factor PAX4 promotes β-cell proliferation and survival in rat and human islets. Journal Cell Biology 167 1123–1135.

    • Search Google Scholar
    • Export Citation
  • Cozar-Castellano I, Takane KK, Bottino R, Balamurugan AN & Stewart AF 2004 Induction of beta-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1. Diabetes 53 149–159.

    • Search Google Scholar
    • Export Citation
  • Csete ME, Benhamou PY, Drazan KE, Wu L, McIntee DF, Afra R, Mullen Y, Busuttil RW & Shaked A 1995 Efficient gene transfer to pancreatic islets mediated by adenoviral vectors. Transplantation 59 263–268.

    • Search Google Scholar
    • Export Citation
  • Diao J, Asghar Z, Chan CB & Wheeler MB 2005 Glucose-regulated glucagon secretion requires insulin receptor expression in pancreatic α-cells. Journal of Biological Chemistry 280 33487–33496.

    • Search Google Scholar
    • Export Citation
  • Diraison F, Parton L, Ferre P, Foufelle F, Briscoe CP, Leclerc I & Rutter GA 2004 Over-expression of sterol-regulatory-element-binding protein-1c (SREBP1c) in rat pancreatic islets induces lipogenesis and decreases glucose-stimulated insulin release: modulation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). Biochemical Journal 378 769–778.

    • Search Google Scholar
    • Export Citation
  • Fernandes JR, Duvivier-Kali VF, Keegan M, Hollister-Lock J, Omer A, Su S, Bonner-Weir S, Feng S, Lee JS, Mulligan RC et al. 2004 Transplantation of islets transduced with CTLA4-Ig and TGFβ using adenovirus and lentivirus vectors. Transplant Immunology 13 191–200.

    • Search Google Scholar
    • Export Citation
  • Garcia-Ocana A, Takane KK, Reddy VT, Lopez-Talavera JC, Vasavada RC & Stewart AF 2003 Adenovirus-mediated hepatocyte growth factor expression in mouse islets improves pancreatic islet transplant performance and reduces beta cell death. Journal of Biological Chemistry 278 343–351.

    • Search Google Scholar
    • Export Citation
  • Giannoukakis N, Rudert WA, Ghivizzani SC, Gambotto A, Ricordi C, Trucco M & Robbins PD 1999 Adenoviral gene transfer of the interleukin-1 receptor antagonist protein to human islets prevents IL-1beta-induced beta-cell impairment and activation of islet cell apoptosis in vitro. Diabetes 48 1730–1736.

    • Search Google Scholar
    • Export Citation
  • Giuliani M, Moritz W, Bodmer E, Dindo D, Kugelmeier P, Lehmann R, Gassmann M, Groscurth P & Weber M 2005 Central necrosis in isolated hypoxic human pancreatic islets: evidence for postisolation ischemia. Cell Transplantation 14 67–76.

    • Search Google Scholar
    • Export Citation
  • Huang XW, Tang ZY, Lawrence TS & Zhang M 2005 5-Fluorouracil and hydroxyurea enhance adenovirus-mediated transgene expression in colon and hepatocellular carcinoma cells. Journal of Cancer Research and Clinical Oncology 131 184–190.

    • Search Google Scholar
    • Export Citation
  • Ilieva A, Yuan S, Wang RN, Agapitos D, Hill DJ & Rosenberg L 1999 Pancreatic islet cell survival following islet isolation: the role of cellular interactions in the pancreas. Journal of Endocrinology 161 357–364.

    • Search Google Scholar
    • Export Citation
  • Irminger JC, Meyer K & Halban PA 1996 Proinsulin processing in the rat insulinoma cell line INS after overexpression of the endoproteases PC2 or PC3 by recombinant adenovirus. Biochemical Journal 320 11–16.

    • Search Google Scholar
    • Export Citation
  • Ishihara H, Maechler P, Gjinovci A, Herrera PL & Wollheim CB 2003 Islet beta-cell secretion determines glucagon release from neighbouring alpha-cells. Nature Cell Biology 5 330–335.

    • Search Google Scholar
    • Export Citation
  • Jornot L, Morris MA, Petersen H, Moix I & Rochat T 2002 N-acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry. Journal of Gene Medicine 4 54–65.

    • Search Google Scholar
    • Export Citation
  • Leclerc I, Woltersdorf WW, da Silva Xavier G, Rowe RL, Cross SE, Korbutt GS, Rajotte RV, Smith R & Rutter GA 2004 Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion. American Journal of Physiology: Endocrinology and Metabolism 286 E1023–E1031.

    • Search Google Scholar
    • Export Citation
  • Leibowitz G, Beattie GM, Kafri T, Cirulli V, Lopez AD, Hayek A & Levine F 1999 Gene transfer to human pancreatic endocrine cells using viral vectors. Diabetes 48 745–753.

    • Search Google Scholar
    • Export Citation
  • Maizel JV Jr, White DO & Scharff MD 1968 The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology 36 115–125.

    • Search Google Scholar
    • Export Citation
  • Miyake S, Makimura M, Kanegae Y, Harada S, Sato Y, Takamori K, Tokuda C & Saito I 1996 Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. PNAS 93 1320–1324.

    • Search Google Scholar
    • Export Citation
  • Narushima M, Okitsu T, Miki A, Yong C, Kobayashi K, Yonekawa Y, Tanaka K, Ikeda H, Matsumoto S, Tanaka N et al. 2004 Adenovirus mediated gene transduction of primarily isolated mouse islets. ASAIO Journal 50 586–590.

    • Search Google Scholar
    • Export Citation
  • Niwa H, Yamamura K & Miyazaki J 1991 Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108 193–199.

    • Search Google Scholar
    • Export Citation
  • Rao P, Roccisana J, Takane KK, Bottino R, Zhao A, Trucco M & Garcia-Ocana A 2005 Gene transfer of constitutively active Akt markedly improves human islet transplant outcomes in diabetic severe combined immunodeficient mice. Diabetes 54 1664–1675.

    • Search Google Scholar
    • Export Citation
  • Takahashi R, Ishihara H, Tamura A, Yamaguchi S, Yamada T, Takei D, Katagiri H, Endou H & Oka Y 2006 Cell type-specific activation of metabolism reveals that β-cell secretion suppresses glucagon release from α-cells in rat pancreatic islets. American Journal of Physiology: Endocrinology and Metabolism 290 E308–E316.

    • Search Google Scholar
    • Export Citation
  • Tran PO, Parker SM, LeRoy E, Franklin CC, Kavanagh TJ, Zhang T, Zhou H, Vliet P, Oseid E, Harmon JS et al. 2004 Adenoviral overexpression of the glutamylcysteine ligase catalytic subunit protects pancreatic islets against oxidative stress. Journal of Biological Chemistry 279 53988–53993.

    • Search Google Scholar
    • Export Citation
  • Triplett JW, Herring BP & Pavalko FM 2005 Adenoviral transgene expression enhanced by cotreatment with etoposide in cultured cells. Biotechniques 39 826–830.

    • Search Google Scholar
    • Export Citation
  • Wang MY, Koyama K, Shimabukuro M, Newgard CB & Unger RH 1998 OB-RB gene transfer to leptin-resistant islets reverses diabetogenic phenotype. PNAS 95 714–718.

    • Search Google Scholar
    • Export Citation
  • Weber M, Deng S, Kucher T, Shaked A, Ketchum RJ & Brayman KL 1997 Adenoviral transfection of isolated pancreatic islets: a study of programmed cell death (apoptosis) and islet function. Journal of Surgical Research 69 23–32.

    • Search Google Scholar
    • Export Citation
  • Yechoor V & Chan L 2005 Gene therapy progress and prospects: gene therapy for diabetes mellitus. Gene Therapy 12 101–107.

  • Zhou YP, Marlen K, Palma JF, Schweitzer A, Reilly L, Gregoire FM, Xu GG, Blume JE & Johnson JD 2003 Overexpression of repressive cAMP response element modulators in high glucose and fatty acid-treated rat islets. A common mechanism for glucose toxicity and lipotoxicity? Journal of Biological Chemistry 278 51316–51323.

    • Search Google Scholar
    • Export Citation

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    Arterial delivery of recombinant adenovirus into mouse pancreas. Polyethylene tubes 0.3 mm in diameter were inserted into the celiac artery (CA, triangle) and the superior mesenteric artery (SMA, arrowhead) at the points branching from the aorta. The pancreas (P) was infused with virus solution and thereby distended. Sp, spleen; St, stomach; L, liver.

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    Adenovirus-mediated eGFP expression in mouse islets. (A) Mouse islets were infected with AdCMV-eGFP immediately after isolation at an m.o.i. of 100, 300, or 900. Percentages of eGFP-positive cells were analyzed by FACS 48 h after infection. Data are means ± s.d. of three independent experiments. (B–D) Immunostaining of sections of mouse islets infected with AdCMV-eGFP (300 m.o.i.) immediately after isolation. Sections of islets were directly observed for eGFP at low magnification (×100) (B) or stained with anti-insulin (C) or anti-glucagon (D) and observed at high magnification (×400). (E–G) Immunostaining of sections of mouse islets infected with AdCMV-eGFP (300 m.o.i.) after an overnight culture. Sections of islets were directly observed for eGFP at low magnification (×100) (E) or stained with anti-insulin (F) or anti-glucagon (G) and observed at high magnification (×400). Bars, 100 μm in B and E, and 20 μm in C, D, F and G.

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    Effects of collagenase treatment on AdCMV-eGFP infection in overnight-cultured islets. (A–C) Immunostaining of sections of mouse islets infected with AdCMV-eGFP after collagenase treatment. Sections of islets were directly observed for eGFP at low magnification (×100) (A) or stained with anti-insulin (B) or anti-glucagon (C) and observed at high magnification (×400). Bars, 100 μm in A and 20 μm in B and C. (D) Flow cytometric analysis of eGFP-positive cells in islets infected after collagenase treatment. One representative result from three experiments is presented. (E) Glucose-stimulated insulin secretion from collagenase-treated islets (closed columns) and non-treated control islets (open columns). The value of insulin secretion from control islets at 2.5 mM glucose (6.54 ± 0.72 ng/h per ten islets, n=3) was taken as 100%. Data are means ± s.e.m. from three independent experiments. (F) Glucose-stimulated insulin secretion from islets infected with AdCMV-eGFP after an overnight culture with collagenase treatment (closed columns) and that of control islets, which were neither infected nor treated with collagenase (open columns). The values of insulin secretion from control islets at 2.5 mM glucose (7.01 ± 0.64 ng/h per ten islets, n=3) was taken as 100%. Data are means ± s.e.m. from three independent experiments. *P<0.05, P<0.01.

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    Efficient gene transduction through arterial delivery of adenovirus vectors. (A–C) Immunostaining of sections of mouse islets infected with AdCMV-eGFP delivered through the CA and the SMA. Sections of islets were directly observed for eGFP at low magnification (×100) (A) or stained with anti-insulin (B) or anti-glucagon (C) and observed at high magnification (×400). Bars, 100 μm in A and 20 μm in B and C. (D) Flow cytometric analysis of eGFP-positive cells in islets infected with AdCMV-eGFP delivered through the CA and the SMA. One representative result out of three is shown. (E) Glucose-stimulated insulin secretion from islets infected with AdCMV-eGFP delivered through the CA and the SMA (closed columns) and that of non-infected islets (open columns). The value of insulin secretion from control islets at 2.5 mM glucose (2.89 ± 0.68 ng/h per ten islets, n=3) was taken as 100%. Data are means ± s.e.m. from three independent experiments.

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    Impact of efficient gene expression, by arterial delivery of recombinant adenovirus, on insulin secretion. (A) Islets infected with AdRIP-HArGlyK immediately after isolation were cultured for 48 h, dispersed and stained with anti-HA or anti-insulin. Experiments were repeated thrice with essentially similar results. (B) Glucose (Glc; 20 mM) or glycerol (Gly; 10 mM)-stimulated insulin secretion from islets infected with AdRIP-HArGlyK immediately after isolation (closed columns) and non-infected control islets (open columns). The value of insulin secretion from control islets at 2.5 mM glucose (6.30 ± 0.77 ng/h per ten islets, n=4) was taken as 100%. Data are means ± s.e.m. from four independent experiments. (C) Islets infected with AdRIP-HArGlyK, delivered through the CA and the SMA, were cultured for 48 h, dispersed and stained with anti-HA or anti-insulin. Experiments were repeated twice with essentially similar results. (D) Glucose (20 mM) or glycerol (10 mM)-stimulated insulin secretion from islets infected with arterially delivered AdRIP-HArGlyK (closed columns) and non-infected control islets (open columns). The value of insulin secretion from control islets at 2.5 mM glucose (3.51 ± 0.63 ng/h per ten islets, n=3) was taken as 100%. Data are means ± s.e.m. from three independent experiments. *P<0.05.

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    Doxycycline-inducible eGFP expression via adenoviruses delivered through the CA and the SMA. AdCAG-TR (0.5×108 pfu) and AdCTO-eGFP (1.5×108 pfu) were injected into the pancreas through the CA and the SMA. Expression of eGFP was induced by treatment with doxycycline (DOX, 2 μg/ml) for 48 h. The pictures shown are representative of three independent experiments. Bars, 20 μm.

  • Bain JR, Schisler JC, Takeuchi K, Newgard CB & Becker TC 2004 An adenovirus vector for efficient RNA interference-mediated suppression of target genes in insulinoma cells and pancreatic islets of Langerhans. Diabetes 53 2190–2194.

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    • Export Citation
  • Barbu AR, Akusjarvi G & Welsh N 2005 Adenoviral-mediated transduction of human pancreatic islets: importance of adenoviral genome for cell viability and association with a deficient antiviral response. Endocrinology 146 2406–2414.

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    • Export Citation
  • Becker TC, BeltrandelRio H, Noel RJ, Johnson JH & Newgard CB 1994 Overexpression of hexokinase I in isolated islets of Langerhans via recombinant adenovirus. Enhancement of glucose metabolism and insulin secretion at basal but not stimulatory glucose levels. Journal of Biological Chemistry 269 21234–21238.

    • Search Google Scholar
    • Export Citation
  • Bertera S, Crawford ML, Alexander AM, Papworth GD, Watkins SC, Robbins PD & Trucco M 2003 Gene transfer of manganese superoxide dismutase extends islet graft function in a mouse model of autoimmune diabetes. Diabetes 52 387–393.

    • Search Google Scholar
    • Export Citation
  • Bilbao G, Contreras JL, Dmitriev I, Smyth CA, Jenkins S, Eckhoff D, Thomas F, Thomas J & Curiel DT 2002 Genetically modified adenovirus vector containing an RGD peptide in the HI loop of the fiber knob improves gene transfer to nonhuman primate isolated pancreatic islets. American Journal of Transplantation 2 237–243.

    • Search Google Scholar
    • Export Citation
  • Brun T, Franklin I, St-Onge L, Biason-Lauber A, Schoenle EJ, Wollheim CB & Gauthier BR 2004 The diabetes-linked transcription factor PAX4 promotes β-cell proliferation and survival in rat and human islets. Journal Cell Biology 167 1123–1135.

    • Search Google Scholar
    • Export Citation
  • Cozar-Castellano I, Takane KK, Bottino R, Balamurugan AN & Stewart AF 2004 Induction of beta-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1. Diabetes 53 149–159.

    • Search Google Scholar
    • Export Citation
  • Csete ME, Benhamou PY, Drazan KE, Wu L, McIntee DF, Afra R, Mullen Y, Busuttil RW & Shaked A 1995 Efficient gene transfer to pancreatic islets mediated by adenoviral vectors. Transplantation 59 263–268.

    • Search Google Scholar
    • Export Citation
  • Diao J, Asghar Z, Chan CB & Wheeler MB 2005 Glucose-regulated glucagon secretion requires insulin receptor expression in pancreatic α-cells. Journal of Biological Chemistry 280 33487–33496.

    • Search Google Scholar
    • Export Citation
  • Diraison F, Parton L, Ferre P, Foufelle F, Briscoe CP, Leclerc I & Rutter GA 2004 Over-expression of sterol-regulatory-element-binding protein-1c (SREBP1c) in rat pancreatic islets induces lipogenesis and decreases glucose-stimulated insulin release: modulation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). Biochemical Journal 378 769–778.

    • Search Google Scholar
    • Export Citation
  • Fernandes JR, Duvivier-Kali VF, Keegan M, Hollister-Lock J, Omer A, Su S, Bonner-Weir S, Feng S, Lee JS, Mulligan RC et al. 2004 Transplantation of islets transduced with CTLA4-Ig and TGFβ using adenovirus and lentivirus vectors. Transplant Immunology 13 191–200.

    • Search Google Scholar
    • Export Citation
  • Garcia-Ocana A, Takane KK, Reddy VT, Lopez-Talavera JC, Vasavada RC & Stewart AF 2003 Adenovirus-mediated hepatocyte growth factor expression in mouse islets improves pancreatic islet transplant performance and reduces beta cell death. Journal of Biological Chemistry 278 343–351.

    • Search Google Scholar
    • Export Citation
  • Giannoukakis N, Rudert WA, Ghivizzani SC, Gambotto A, Ricordi C, Trucco M & Robbins PD 1999 Adenoviral gene transfer of the interleukin-1 receptor antagonist protein to human islets prevents IL-1beta-induced beta-cell impairment and activation of islet cell apoptosis in vitro. Diabetes 48 1730–1736.

    • Search Google Scholar
    • Export Citation
  • Giuliani M, Moritz W, Bodmer E, Dindo D, Kugelmeier P, Lehmann R, Gassmann M, Groscurth P & Weber M 2005 Central necrosis in isolated hypoxic human pancreatic islets: evidence for postisolation ischemia. Cell Transplantation 14 67–76.

    • Search Google Scholar
    • Export Citation
  • Huang XW, Tang ZY, Lawrence TS & Zhang M 2005 5-Fluorouracil and hydroxyurea enhance adenovirus-mediated transgene expression in colon and hepatocellular carcinoma cells. Journal of Cancer Research and Clinical Oncology 131 184–190.

    • Search Google Scholar
    • Export Citation
  • Ilieva A, Yuan S, Wang RN, Agapitos D, Hill DJ & Rosenberg L 1999 Pancreatic islet cell survival following islet isolation: the role of cellular interactions in the pancreas. Journal of Endocrinology 161 357–364.

    • Search Google Scholar
    • Export Citation
  • Irminger JC, Meyer K & Halban PA 1996 Proinsulin processing in the rat insulinoma cell line INS after overexpression of the endoproteases PC2 or PC3 by recombinant adenovirus. Biochemical Journal 320 11–16.

    • Search Google Scholar
    • Export Citation
  • Ishihara H, Maechler P, Gjinovci A, Herrera PL & Wollheim CB 2003 Islet beta-cell secretion determines glucagon release from neighbouring alpha-cells. Nature Cell Biology 5 330–335.

    • Search Google Scholar
    • Export Citation
  • Jornot L, Morris MA, Petersen H, Moix I & Rochat T 2002 N-acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry. Journal of Gene Medicine 4 54–65.

    • Search Google Scholar
    • Export Citation
  • Leclerc I, Woltersdorf WW, da Silva Xavier G, Rowe RL, Cross SE, Korbutt GS, Rajotte RV, Smith R & Rutter GA 2004 Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion. American Journal of Physiology: Endocrinology and Metabolism 286 E1023–E1031.

    • Search Google Scholar
    • Export Citation
  • Leibowitz G, Beattie GM, Kafri T, Cirulli V, Lopez AD, Hayek A & Levine F 1999 Gene transfer to human pancreatic endocrine cells using viral vectors. Diabetes 48 745–753.

    • Search Google Scholar
    • Export Citation
  • Maizel JV Jr, White DO & Scharff MD 1968 The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology 36 115–125.

    • Search Google Scholar
    • Export Citation
  • Miyake S, Makimura M, Kanegae Y, Harada S, Sato Y, Takamori K, Tokuda C & Saito I 1996 Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. PNAS 93 1320–1324.

    • Search Google Scholar
    • Export Citation
  • Narushima M, Okitsu T, Miki A, Yong C, Kobayashi K, Yonekawa Y, Tanaka K, Ikeda H, Matsumoto S, Tanaka N et al. 2004 Adenovirus mediated gene transduction of primarily isolated mouse islets. ASAIO Journal 50 586–590.

    • Search Google Scholar
    • Export Citation
  • Niwa H, Yamamura K & Miyazaki J 1991 Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108 193–199.

    • Search Google Scholar
    • Export Citation
  • Rao P, Roccisana J, Takane KK, Bottino R, Zhao A, Trucco M & Garcia-Ocana A 2005 Gene transfer of constitutively active Akt markedly improves human islet transplant outcomes in diabetic severe combined immunodeficient mice. Diabetes 54 1664–1675.

    • Search Google Scholar
    • Export Citation
  • Takahashi R, Ishihara H, Tamura A, Yamaguchi S, Yamada T, Takei D, Katagiri H, Endou H & Oka Y 2006 Cell type-specific activation of metabolism reveals that β-cell secretion suppresses glucagon release from α-cells in rat pancreatic islets. American Journal of Physiology: Endocrinology and Metabolism 290 E308–E316.

    • Search Google Scholar
    • Export Citation
  • Tran PO, Parker SM, LeRoy E, Franklin CC, Kavanagh TJ, Zhang T, Zhou H, Vliet P, Oseid E, Harmon JS et al. 2004 Adenoviral overexpression of the glutamylcysteine ligase catalytic subunit protects pancreatic islets against oxidative stress. Journal of Biological Chemistry 279 53988–53993.

    • Search Google Scholar
    • Export Citation
  • Triplett JW, Herring BP & Pavalko FM 2005 Adenoviral transgene expression enhanced by cotreatment with etoposide in cultured cells. Biotechniques 39 826–830.

    • Search Google Scholar
    • Export Citation
  • Wang MY, Koyama K, Shimabukuro M, Newgard CB & Unger RH 1998 OB-RB gene transfer to leptin-resistant islets reverses diabetogenic phenotype. PNAS 95 714–718.

    • Search Google Scholar
    • Export Citation
  • Weber M, Deng S, Kucher T, Shaked A, Ketchum RJ & Brayman KL 1997 Adenoviral transfection of isolated pancreatic islets: a study of programmed cell death (apoptosis) and islet function. Journal of Surgical Research 69 23–32.

    • Search Google Scholar
    • Export Citation
  • Yechoor V & Chan L 2005 Gene therapy progress and prospects: gene therapy for diabetes mellitus. Gene Therapy 12 101–107.

  • Zhou YP, Marlen K, Palma JF, Schweitzer A, Reilly L, Gregoire FM, Xu GG, Blume JE & Johnson JD 2003 Overexpression of repressive cAMP response element modulators in high glucose and fatty acid-treated rat islets. A common mechanism for glucose toxicity and lipotoxicity? Journal of Biological Chemistry 278 51316–51323.

    • Search Google Scholar
    • Export Citation