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).
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.
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.
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.
The authors thank the National Institutes of Health Islet Cell Resource, and the Basic Science Islet Distribution Program for providing them with human islets.
1. Dor Y, Brown J, Martinez OI, et al. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature
2004; 429: 41.
2. Meier JJ, Lin JC, Butler AE, et al. Direct evidence of attempted beta cell regeneration in an 89-year-old patient with recent-onset type 1 diabetes
2006; 49: 1838.
3. Hardikar AA. Generating new pancreas from old. Trends Endocrinol Metab
2004; 15: 198.
4. Cozar-Castellano I, Takane KK, Bottino R, et al. 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
2004; 53: 149.
5. Rao P, Roccisana J, Takane KK, et al. Gene transfer of constitutively active Akt markedly improves human islet transplant outcomes in diabetic severe combined immunodeficient mice. Diabetes
2005; 54: 1664.
6. Contreras JL, Bilbao G, Smyth C, et al. Gene transfer of the Bcl-2 gene confers cytoprotection to isolated adult porcine pancreatic islets exposed to xenoreactive antibodies and complement. Surgery
2001; 130: 166.
7. Emamaullee JA, Rajotte RV, Liston P, et al. XIAP overexpression in human islets prevents early posttransplant apoptosis and reduces the islet mass needed to treat diabetes
2005; 54: 2541.
8. Fenjves ES, Ochoa MS, Gay-Rabinstein C, et al. Adenoviral gene transfer of erythropoietin confers cytoprotection to isolated pancreatic islets. Transplantation
2004; 77: 13.
9. Heimberg H, Heremans Y, Jobin C, et al. Inhibition of cytokine-induced NF-kappaB activation by adenovirus-mediated expression of a NF-kappaB super-repressor prevents beta-cell apoptosis. Diabetes
2001; 50: 2219.
10. Fiaschi-Taesch NM, Berman DM, Sicari BM, et al. Hepatocyte growth factor enhances engraftment and function of nonhuman primate islets. Diabetes
2008; 57: 2745.
11. Plesner A, Liston P, Tan R, et al. The X-linked inhibitor of apoptosis protein enhances survival of murine islet allografts. Diabetes
2005; 54: 2533.
12. Noguchi H, Xu G, Matsumoto S, et al. Induction of pancreatic stem/progenitor cells into insulin-producing cells by adenoviral-mediated gene transfer technology. Cell Transplant
2006; 15: 929.
13. Schisler JC, Fueger PT, Babu DA, et al. Stimulation of human and rat islet beta-cell proliferation with retention of function by the homeodomain transcription factor Nkx6.1. Mol Cell Biol
2008; 28: 3465.
14. Le LP, Le HN, Dmitriev IP, et al. Dynamic monitoring of oncolytic adenovirus in vivo by genetic capsid labeling. J Natl Cancer Inst
2006; 98: 203.
15. Hanada M, Feng J, Hemmings BA. Structure, regulation and function of PKB/AKT–a major therapeutic target. Biochim Biophys Acta
2004; 1697: 3.
16. Besson A, Dowdy SF, Roberts JM. CDK inhibitors: Cell cycle regulators and beyond. Dev Cell
2008; 14: 159.
17. Heron-Milhavet L, Franckhauser C, Rana V, et al. Only Akt1 is required for proliferation, while Akt2 promotes cell cycle exit through p21 binding. Mol Cell Biol
2006; 26: 8267.
18. Manning BD, Cantley LC. AKT/PKB signaling: Navigating downstream. Cell
2007; 129: 1261.
19. Fatrai S, Elghazi L, Balcazar N, et al. Akt induces beta-cell proliferation by regulating cyclin D1, cyclin D2, and p21 levels and cyclin-dependent kinase-4 activity. Diabetes
2006; 55: 318.
20. Jetton TL, Lausier J, LaRock K, et al. Mechanisms of compensatory beta-cell growth in insulin-resistant rats: Roles of Akt kinase. Diabetes
2005; 54: 2294.
21. Tuttle RL, Gill NS, Pugh W, et al. Regulation of pancreatic beta-cell growth and survival by the serine/threonine protein kinase Akt1/PKBalpha. Nat Med
2001; 7: 1133.
22. Bernal-Mizrachi E, Wen W, Stahlhut S, et al. Islet beta cell expression of constitutively active Akt1/PKB alpha induces striking hypertrophy, hyperplasia, and hyperinsulinemia. J Clin Invest
2001; 108: 1631.
23. Contreras JL, Smyth CA, Bilbao G, et al. Simvastatin induces activation of the serine-threonine protein kinase AKT and increases survival of isolated human pancreatic islets. Transplantation
2002; 74: 1063.