Adenovirus Infection Activates Akt1 and Induces Cell Proliferation in Pancreatic Islets1 : Transplantation

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Basic and Experimental Research

Adenovirus Infection Activates Akt1 and Induces Cell Proliferation in Pancreatic Islets1

Icyuz, Mert1; Bryant, Stacie M.J.3; Fortinberry, Henry K.3; Molakandov, Kfir1,4; Siegal, Gene P.5,6; Contreras, Juan L.3; Wu, Hongju1,2,7

Author Information
Transplantation 87(6):p 821-824, March 27, 2009. | DOI: 10.1097/TP.0b013e318199c686
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Abstract

Diabetes mellitus is a common chronic disease whose origin is believed to involve the disturbance of insulin secretion and insulin signaling pathways, resulting in inadequate homeostatic control of blood glucose. The key pancreatic tissue responsible for blood glucose homeostasis is the endocrine cell population known as the islets of Langerhans in which insulin is produced by the β cells and glucagon by the α cells. As terminally differentiated cells, proliferation is not expected in the islet cells in adulthood. Nonetheless, in recent years, accumulated evidence has shown that pancreatic β cells have the potential to proliferate, although limited in scope (1, 2). This potential for β-cell proliferation is enhanced under a variety of circumstances including pancreatectomy, cellophane wrapping, and bone marrow transplantation (3).

Human adenoviruses serotype 5 (Ad5), have been used to deliver genes of interest into pancreatic islets to protect them from damage and to investigate the role of particular genes of interest in pancreatic islet cell differentiation (4, 5). For example, Ad5 has been used to transfer cytokine signaling blocking genes and antiapoptotic genes into donor islets in studies aiming at improving the efficacy of islet transplantation (5–11). Ad5 is also a common gene delivery vector to elucidate the roles of transcription factors, such as Pdx1, Ngn3, and Nkx6.1, in islet cell differentiation and regeneration (12, 13).

This study attempted to assess whether Ad5 infection had any impact on the potential of β-cell proliferation. Ad5 encoding a rat insulin promoter (RIP)-driven firefly luciferase (Luc) or green fluorescence protein (GFP), namely Ad5.RIP-Luc and Ad5.RIP-GFP, were used in the study. Initially, we examined the expression of several key proliferation molecules in the uninfected or infected rat and pig islets (Fig. 1). The freshly isolated rat or pig islets were infected with Ad5.RIP-Luc or Ad5.RIP-GFP at a multiplicity of infection of 500 viral particles per cell (VPs/cell). Ad5.RIP-Luc infection of the islets were demonstrated by Luc bioluminescence imaging (14) (Fig. 1A, upper) and Ad5.RIP-GFP infection by GFP microscopy (Fig. 1A, lower). Western blotting assays were performed 2 days after the viral infection. As shown in Figure 1(B), we found both Ad5.RIP-Luc and Ad5.RIP-GFP induced the expression of Akt1, a serine/threonine protein kinase B (PKB), in both rat and pig islets. Akt1 became readily detectable upon Ad5 infection with an antibody recognizing the C-terminal region of human Akt1 (C-20). Similar results were obtained with Ad5 vectors carrying other transgenes (data not shown).

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FIGURE 1.:
Human adenovirus serotype 5 (Ad5) infection induced Akt1 expression and phosphorylation in freshly isolated rat and pig islets. (A) Firefly luciferase (Luc) bioluminescence imaging or green fluorescence protein (GFP) microscopy showing Ad5 infection in rat islets. The freshly isolated rat islets were plated in 24-well plates at a density of 150 IEQs/well, and infected with Ad5.rat insulin promoter (RIP)-Luc or Ad5.RIP-GFP at a multiplicity of infection of 500 viral particles/cell. Uninfected islets were processed in parallel as a control. At various days postinfection, noninvasive Luc imaging was performed for Ad5.RIP-Luc infected islets with a custom-built noninvasive optical imaging system (14). Fluorescence microscopy for Ad5.RIP-GFP infected islets was performed 2 days postinfection. (B) Western blotting analysis of Akt1 expression and phosphorylation in rat and pig islets. Two days after Ad5 infection (multiplicity of infection=500 viral particles/cell), the islets were lysed and processed for western blotting analysis using C-20 (to detect total Akt1) (Santa Curz Biotechnology, CA, USA) and antibodies recognizing phos-Thr308 and phos-Ser473 (Cell Signaling Technology, Danvers, MA, USA). Lane 1: uninfected; lane 2: Ad5.RIP-Luc infected; and lane 3: Ad5.RIP-GFP infected. Representative data from three independent experiments are shown.

As the direct downstream target of phosphoinositide 3 kinase (PI3K), Akt1 is a key player in the PI3K signal transduction pathway that is activated in response to growth factors or insulin (15). Activation of PI3K leads to the generation of phosphatidylinositol trisphosphate, which binds Akt1 and induces its translocation to the plasma membrane where Akt1 is activated by phosphorylation at two residues, Thr308 and Ser473. We thus examined whether the induced Akt1 is indeed active (phosphorylated). By using antibodies specifically recognizing the phosphorylated sites, phos-Thr308 and phos-Ser473, we found Akt1 was phosphorylated at Ser473, but not at Thr308 (Fig. 1B). Of note, both phos-Thr308 and phos-Thr473 antibodies were raised against mouse Akt1 peptides. Therefore, our inability to detect phos-Thr308 may be attributable to low level of phos-Thr308, or because of the failure of anti-mouse phos-Thr308 to recognize the rat and pig proteins.

To further explore this matter, we investigated the effect of Ad5 infection on human islets. The freshly isolated human islets (≥90% viability and ≥85% purity) were obtained from the National Institutes of Health Islet Cell Resource through the Islet Cell Resource Basic Science Islet Distribution Program, and infected with Ad5.RIP-Luc (Fig. 2). We found Ad5.RIP-Luc infection induced expression and phosphorylation of Akt1 in human islets (Fig. 2B). Interestingly, Akt1 was found to be phosphorylated at both sites, although phosphorylation at Thr308 seemed to be weaker than that at Ser473. It should be noted that we have repeatedly observed more robust expression and phosphorylation of Akt1 in Ad5-infected human islets than in rat and pig islets with the same amount of vector and islets. This may be explained by better recognition of the antibodies for human Akt1 and phos-Akt1 than for rat and pig proteins, although the phos-Akt1 was raised against mouse Akt1 peptides.

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FIGURE 2.:
Western blotting assays examining the expression of key proteins involved in cell proliferation in human islets. The human islets were infected with human adenovirus serotype 5 (Ad5).rat insulin promoter-firefly luciferase (Luc) (lane: Ad5) at a multiplicity of infection of 500 viral particles/cell. Uninfected islets (lane: un) were used as control. Two days later, the islets were processed for biochemical Luc assay using a commercial kit (Promega, Madison, WI) or western blotting analysis using specific antibodies. (A) Infectivity of Ad5.rat insulin promoter-Luc in human islets as indicated by Luc activity. The relative light units representing Luc activity were normalized with the total amount of protein in the lysates. (B) Expression and activation of Akt1 in human islets. (C) Expression of cyclin D3, cyclin E, p21 and p27 in human islets. All of the antibodies were purchased from Cell Signaling Technology, Inc., (Danvers, MA). The β-actin staining was included to show same amount of proteins was loaded in the uninfected and Ad5 lanes. Presented are representative data from four independent experiments.

Akt1 activation has been shown to stimulate proliferation through multiple downstream targets involved in cell-cycle regulation including cyclins and cyclin-dependent kinase (CDK) inhibitors (16–18). Previous studies in transgenic mice suggest Akt1 may induce β-cell proliferation by regulating cyclin D1, D2, and p21 (19). We, thus, examined whether the expression of these proteins was changed by Ad5 infection. The expression of cyclin A, cyclin D1 and D2 in uninfected or Ad5-infected human islets was not detectable by western blotting assays under the experimental conditions (data not shown). Nonetheless, we found cyclin D3, but not cyclin E, was significantly up-regulated by Ad5 infection (Fig. 2C). In addition, the expression of p21, a member of Cip/Kip family of CDK inhibitors, was up-regulated. In contrast, the expression of another Cip/Kip family protein p27 was readily detectable, but did not seem to be affected by Ad5 infection (Fig. 2C).

Akt1 is a key mediator of the growth factors that control cell cycle progression and cell survival. We, therefore, examined whether Ad5 infection could induce β-cell proliferation in purified human islets. In these experiments, 5-Bromo-2′-deoxy-uridine (BrdU), a thymidine analog, was used to label proliferating cells because it can be incorporated into the newly synthesized genome during DNA replication. The proliferating cells were identified by immunofluorescence staining with anti-BrdU antibody. Insulin (to identify β-cells) antibody and Hoechst staining (for nuclei) were also included.

As shown in Figure 3, BrdU+ β-cells (BrdU+/Insulin+) in uninfected human islets were hardly detectable (only one positive β-cell in 59 islets), suggesting spontaneous β-cell proliferation occurs rarely. In contrast, BrdU+ β-cells were detected with much higher frequency in the islets infected with Ad5.RIP-Luc (13 BrdU+ β-cells in 57 islets). Figure 3(A) shows representative confocal microscope images. A proliferating β-cell (BrdU+/Insulin+) was identified in an Ad5.RIP-Luc-infected human islet (marked by arrow). Summarized in Figure 3(B) is the representative experiment showing the average number of BrdU+ β-cells per islet, and the percentage of islets containing BrdU+ β-cells versus the total islets. Taken together, these data demonstrated that Ad5 infection induced β-cell proliferation in pancreatic islets, which is consistent with our western blotting data showing Ad5 infection resulted in endogenous Akt1 expression and activation.

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FIGURE 3.:
Human adenovirus serotype 5 infection promoted β-cell proliferation in human islets in vitro as assessed by 5-Bromo-2′-deoxy-uridine (BrdU) incorporation. Freshly isolated human islets were infected with human adenovirus serotype 5.rat insulin promoter-Luc at multiplicity of infection of 250 viral particles/cell and incubated in the presence of BrdU (25 μg/mL) for 4 days in vitro. Uninfected human islets were processed in parallel as a control. The islets were fixed, embedded, sectioned, and processed for immunofluorescence staining. (A) Representative images obtained by confocal microscopy. (B) Summary of the distribution of BrdU+ β-cells in each infection group. (A) The average number of BrdU+ cells per islet was calculated by dividing the total BrdU+ β-cells by the total number of islets in each group. (B) The percentage of islets containing BrdU+ β-cells was calculated by dividing the islets that contained one or more BrdU+ β-cells by the total number of islets in each group. Data shown are representatives of three independent experiments.

This study assessed the impact of Ad5 infection on β-cell proliferation. We found that Ad5 infection induced the expression and activation of Akt1, a key mediator of the PI3K signaling pathway and known to have a profound cell survival and proliferating effect. It should be noted that, although only the data on Ad5 carrying reporter genes were included here, similar results were obtained with other Ad5 vectors (encoding different functional transgenes) as well (data not shown). The induced Akt1 expression and activation correlate with the enhanced β-cell proliferation in Ad5-infected human islets.

How Akt1 is activated by Ad5 is not clear. It has been demonstrated that Akt1 activation occurs mainly through the PI3K signaling pathway (15). Binding of ligands (such as growth factors and insulin) to the receptor tyrosine kinases leads to activation of PI3K, which, in turn, result in phosphorylation and activation of Akt1. This process is regulated by several “regulatory proteins” and proteins involved in other intracellular signal transduction pathways (15). Whether Ad5 infection induces Akt1 expression and activation by acting on the receptor or on the regulatory proteins remains to be investigated.

Previous studies have suggested that Akt1 is essential for β-cell growth and proliferation (19–21). In particular, transgenic mice overexpressing active Akt1 have shown significant increase in islet mass and are resistant to streptozotocin-induced diabetes (21, 22). In fact, the relevance of Akt1 in β-cell proliferation has been explored as a means to improve the therapeutic outcome of islet transplantation (5, 23). Furthermore, it has been suggested that Akt1-induced β-cell proliferation acts through cyclin D1, cyclin D2, CDK4, and p21 (19). This study attempted to assess the expression of these and several other proteins involved in cell cycle, survival and proliferation events. We found p21 was up-regulated in Ad5 infected human islets, which is consistent with the previous studies using Akt1 transgenic mice (19). However, we failed to detect cyclin D1 and D2 by western blotting assays, probably because of the low expression levels of these proteins under the experimental conditions. Interestingly, cyclin D3 seemed to be significantly up-regulated by Ad5 infection. The discrepancy, however, is consistent with the concept that different Akt1-activating stimuli lead to recruitment of selective Akt1 downstream targets, and result in stimuli-specific cellular functions (18).

Ad5 vectors have been used to deliver genes of interest into pancreatic islets. This is the first study showing that Ad5 infection by itself can induce the expression and activation of endogenous Akt1, which in turn promotes β-cell proliferation. This information has significant ramification for studies involving Ad5-mediated gene delivery into pancreatic islet cells.

ACKNOWLEDGMENTS

The authors thank the National Institutes of Health Islet Cell Resource, and the Basic Science Islet Distribution Program for providing them with human islets.

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Keywords:

Adenovirus; Akt1; β-Cell proliferation; Diabetes

© 2009 Lippincott Williams & Wilkins, Inc.