Glucose drives insulin secretion and promotes rodent beta cell proliferation and survival (1,2). Several growth factors including insulin-like growth factor-I (IGF-I), glucagon-like peptide 1 (GLP-1) and hepatocyte growth factor (HGF) have also been shown to increase proliferation, enhance function and/or promote survival of the pancreatic beta cell (2-11). Activation of the phosphatidylinositol 3’-kinase (PI3K)-Protein-Kinase B (PKB)/Akt signaling pathway by glucose and the aforementioned growth factors seems to be of importance for the enhancement of beta cell proliferation and survival induced by these agonists (1-3,6,7,10). Moreover, beta cell-specific expression of a constitutively active form of PKB/Akt in transgenic mice markedly increases beta cell mass resulting in hyperinsulinemia and hypoglycemia (12,13). Taken together, these findings make PKB/Akt an attractive intracellular target for therapeutic approaches in enhancing human islet expansion in vitro and improving human islet transplant outcomes.
Intracellular signaling through the PKB/Akt pathway: Targets and actions
In this section, the intention is to briefly describe the actions and intracellular signal transduction targets of PKB/Akt. For a more detailed description, excellent reviews have been written over the last five years that provide a more complete and extensive picture of PKB/Akt functions and interactions (14-17). The three main isoforms of PKB/Akt identified in mammalian cells (PKBα/Akt1, PKBß/Akt2 and PKBγ/Akt3) derived from three distinct genes, contain a C-terminal regulatory domain, a catalytic domain and an N-terminal pleckstrin homology (PH) domain that is highly conserved in all three members (Figure 1). Cellular processes such as proliferation, survival, and glucose metabolism induced by different hormones and growth factors are dependent on the activation of PI3-kinase. Activation of PI3-kinase generates D3-phosphorylated phosphoinositides that are capable of binding PKB/Akt through the PH domain (Figures 1 and 2). Recent studies have suggested that the role of the PH domain in PKB/Akt regulation may not be limited to lipid binding, but also to be used as a protein interaction domain.
Figure 2 shows a schematic representation of how PKB/Akt molecule is activated inside the cell. Briefly, generation of D3-phosphorylated phosphoinositides by PI3K induces the translocation of PKB/Akt to the plasma membrane where it is activated. In the plasma membrane, PKB/Akt co-localizes with the constitutively active phosphoinositide-dependent kinase-1 (PDK-1) and another unknown kinase (perhaps PDK2 or PKB/Akt itself) that activates PKB/Akt by phosphorylation of different residues such as Thr308 and Ser473 in the PKBα/Akt1 isoform, respectively. Phosphatases such as PP2A deactivate PKB/Akt. Fusion of N-terminal c-Src myristolation sequences to PKB/Akt (with or without the PH domain) results in a constitutive active form of PKB/Akt (2,12,13,18). In this case, PKB/Akt myristolation directly targets the kinase to the membrane where it is phosphorylated and constitutively activated inside the cell (Figure 1).
PKB/Akt has emerged as the focal point for many intracellular signal transduction pathways, regulating multiple cellular processes such as apoptosis, proliferation, angiogenesis and glucose metabolism (15-17). Among the myriad of downstream substrates or modulators, which could influence PKB/Akt intracellular functions, are BAD, caspase-9, glycogen synthase kinase-3 a/ß (GSK-3a/ß), the family of forkhead box (Fox) transcription factors and murine double minute 2 (MDM2). PKB/Akt has been shown to phosphorylate these proteins impeding them from performing their pro-apoptotic functions. Phosphorylation/activation of some of these proteins such as MDM2, Fox transcription factors, and others such as p27 and Cyclin D (in this case through GSK-3a/ß) have been shown to be also implicated in the proliferative effects of PKB/Akt. PKB/Akt-induced phosphorylation of the endothelial nitric oxide synthase (eNOS) at Ser1179, and consequent activation leads to NO production by the endothelial cells. Nitric oxide production by eNOS is a key regulator of vascular angiogenesis and vascular remodeling after injury. However, the exact mechanism by which PKB/Akt activates/interacts with eNOS is not known.
Activation of PKB/Akt is also a key component of the insulin response in insulin target tissues, mediating diverse aspects of glucose metabolism. Muscle and fat are the main tissues involved in insulin-mediated glucose disposal. These two tissues synthesize the insulin-sensitive glucose transporter isoform, GLUT-4, which redistributes from intracellular storage vesicles to the plasma membrane after insulin stimulation. Insulin mediates the recruitment of PKB/Akt to GLUT-4-containing vesicles where this kinase seems to phosphorylate component proteins of these vesicles. The exact nature of the recruitment of PKB/Akt to these vesicles is unclear. Insulin also promotes protein synthesis in liver and muscle by stimulating the initiation and elongation steps in protein translation. It has been suggested that PKB/Akt might be implicated in the phosphorylation/activation of the mammalian target of rapamycin (mTOR) that then releases the translation initiation factor, eIF-4E, and initiates translation. mTOR phosphorylation by PKB/Akt can lead to phosphorylation/activation of p70-kDa-S6-kinase (p70S6K) that then activates ribosomal S6 protein and also enhances protein translation. Mice deficient in p70S6K display hypoinsulinemia, glucose intolerance and diminished beta cell size (19).
Recently, several laboratories have reported the effects of targeted disruption of PKBα/Akt1 or PKBß/Akt2 in knockout mice (20-22). Interestingly, targeted disruption of PKBα/Akt1 in mice results in a conspicuous impairment in organismal growth with no changes in glucose homeostasis (20). On the other hand, the targeted elimination of PKBß/Akt2 in mice results in a phenotype with similarities to human Type 2 diabetes (21,22). These mice display hyperglycemia, hyperinsulinemia, impaired glucose tolerance, insulin resistance and mild growth retardation. In males, insulin resistance progresses to a severe form of diabetes accompanied by pancreatic beta cell failure (22). Taken together, these studies suggest that PKB/Akt pathway plays a critical role in growth, glucose metabolism as well as in the maintenance of beta cell mass.
PKB/Akt and the rodent pancreatic beta cell
Regarding the presence of PKB/Akt in the pancreatic beta cell, all three isoforms of PKB/Akt have been detected in pancreatic islets and in continuous beta cell lines derived from rodent insulinomas (23). It has been demonstrated that activation of PKB/Akt in beta cells occurs after exposure to growth factors such as IGF-1, HGF and GLP-1 (2,6,10,18,23). Inhibition of PI3K activity and PKB/Akt phosphorylation with wortmannin has been shown to blunt both the proliferative and pro-survival effects induced by HGF in INS-1 cells (7,10). Furthermore, adenoviral expression of a kinase-dead form of PKB in INS-1 cells decreased IGF-1-induced beta-cell proliferation (2). Glucose per se has also been shown to increase the phosphorylation/activation of PKB/Akt in insulinoma cells and mouse islets (1). Transfection of insulinoma cells with the cDNA of a kinase-dead form of PKB/Akt resulted in loss of glucose-induced prosurvival effects in these beta cells (1). Furthermore, transfection with constitutively active PKB/Akt enhanced survival in glucose-deprived insulinoma cells (1). Moreover, glucose- and IGF-I-induced activation of PKB/Akt in INS-1 cells is reduced in the presence of free fatty acids (FFA) (18). Interestingly, this decrease in PKB/Akt activity correlated with an increase in the incidence of FFA-induced apoptosis in these cells (18). Again, adenovirus mediated expression of constitutively active PKB/Akt almost completely prevented FFA-induced apoptosis in INS-1 cells (18). These pro-survival effects of constitutively active PKB/Akt in FFA-treated INS-1 cells are in part mediated by phosphorylation/inactivation of GSK-3a/ß and FoxO1, and inactivation of caspase 9 and p53 (18). Taken together, these in vitro studies indicate that activation of the PI3K-PKB/Akt pathway plays an important role in promoting pancreatic beta cell proliferation and enhancing survival under basal and stress-induced conditions.
Two groups have recently reported the generation of transgenic mice with specific expression of constitutively active PKBα/Akt1 in the pancreatic beta cell (12,13). In both studies, the constitutively active kinase strikingly increased beta cell mass (8- to 10-fold) resulting in hyperinsulinemia and decreased blood glucose levels. The increase in beta cell mass observed in these transgenic mice is the result of an increase in beta cell size (hypertrophy) and probably increased islet neogenesis, a difficult parameter to quantitate. Importantly, in both studies, constitutively active PKB/Akt induced an increase in beta cell survival in vivo after treatment of transgenic mice with the diabetogenic and cytotoxic agent streptozotocin. However, the results regarding beta cell proliferation in these mice are more controversial. While in one of the studies the authors detected an increase in beta cell proliferation in adult mice (13), this effect was not observed in the other study (12). Perhaps differences in mouse strains or in the level of expression of constitutively actyive PKB/Akt in the beta cell could explain this discrepancy. Collectively, these studies indicate that constitutive activation of PKB/Akt in the beta cell promotes hyperinsulinemia, and beta cell hyperplasia and survival in vivo, and strongly support the idea that PKB/Akt itself or downstream signaling targets could be useful for therapeutic intervention in increasing beta cell mass and survival.
Bernal-Mizrachi et al. have recently reported the effect of decreased Akt activity specifically in the beta cell of transgenic mice (24). In these studies, the rat insulin type I promoter was used to drive beta cell overexpression of a kinase-dead Akt1 with a mutation at the ATP binding site that allows this molecule to act in a dominant negative manner. Interestingly, these transgenic mice displayed impaired glucose tolerance, defective insulin secretion and increased susceptibility to develop diabetes following fat-feeding (24). The authors also found that the decrease in Akt activity in the beta cell caused a defect in insulin exocytosis that resulted in dysregulated insulin secretion. These studies are important since they indicate for the first time that PKB/Akt can also play an important role in the regulation of normal beta cell function and suggest that this enzyme is an important therapeutic target for improving insulin secretion in diabetes.
Human islet transplantation
Recent studies by Shapiro and colleagues at the University of Alberta in Edmonton, Canada, have demonstrated that human islet allograft transplantation can be a highly successful (75-80%) therapeutic option in the treatment of patients with Type I diabetes (25-27). This new islet transplantation protocol, known as the Edmonton Protocol, combines the use of low doses of glucocorticoid-free immunosuppressants with a more effective method of islet isolation (shorter cold-ischemia time and use of human albumin) (25-27). However, despite this enormous advance, these studies have highlighted a critical paucity of pancreatic islet donors. Every transplanted patient receives islets from 2 to 4 cadaver donors. Since the number of patients with Type 1 diabetes in the world highly exceeds the number of pancreatic islet donors, there is an obvious mismatch between the demand for, and the availability of, human islets for transplantation. This mismatch has prompted many labs to develop methods and strategies for increasing the availability of beta cells or beta cell precursors that potentially could be transplanted into patients with diabetes. Among these strategies are: (i) islet encapsulation; (ii) conversion of embryonic stem cells or pancreatic progenitor cells into insulin-producing cells; (iii) generation of immortalized human beta cell lines; and (iv) development of ways to increase beta cell proliferation, function and survival in a transplant setting, so that smaller numbers of pancreatic islets could be used per recipient (28).
PKB/Akt and the human islet
Recently, PKB/Akt expression has been detected in isolated human islets (29,30). Wortmannin-mediated inhibition of the PI3K-PKB/Akt pathway in isolated human islets induced the phosphorylation of both apoptosis signal regulating kinase 1 (ASK 1), a kinase upstream of c-jun NH2-terminal kinase (JNK), and JNK. Furthermore, JNK activation led to enhanced basal islet cell death and increased sensitivity to cytokine-mediated cell death (30). These studies indicate that there is a cross-talk between PI3K/Akt and JNK pathways that mediates the survival of isolated human islets. The use of JNK inhibitors or PKB/Akt activators may be a viable strategy for increasing human islet cell survival after isolation.
Contreras et al. have reported important findings regarding the role of PKB/Akt activation in human islet transplant outcomes in diabetic mice (29). They treated isolated human islets in vitro with the cholesterol-lowering drug, simvastatin. Statins have been shown to reduce the primary non-function of pancreatic islet grafts and to activate the PI3K -PKB/Akt pathway (31). Interestingly, human islets treated with simvastatin display Akt activation, increased islet viability, and decreased Bad phosphorylation, cytochrome C release, caspase-9 activation and Fox transcription factor translocation to the nucleus. These effects were reversed with co-treatment with the PI3-kinase inhibitor, LY294002. More importantly, a marginal mass of human islets treated with simvastatin and transplanted in diabetic SCID mice restored euglycemia in 58% of the cases. This effect was completely abolished when islets were pre-treated with the aforementioned PI3-kinase inhibitor. These studies are important since they establish that PI3-kinase-PKB/Akt activation, as a potential therapeutic approach, is applicable to human islet transplantation.
Conclusions
Davalli et al. have demonstrated that the first days after transplantation are crucial for a transplant to be successful (32). Massive cell loss in the islet graft has been reported to occur immediately after transplantation. Protection and expansion of these islet cell grafts may potentially help to reduce the number of islets needed for successful transplantation. Constitutive activation of PKB/Akt in the pancreatic beta cell in rodents induces hyperplasia and resistance to cell death (1,2,12,13,18). These characteristics make PKB/Akt an attractive intracellular target for therapeutic approaches in improving islet transplantation. However, no attempt has been made to determine whether constitutive activation of PKB/Akt in human islet cells could enhance the engraftment and survival of transplanted islets in diabetic SCID mice. Furthermore, does constitutive activation of Akt in human islets enhance beta cell proliferation and expand beta cell mass in vitro? Does constitutive activation of Akt in human islets improve beta cell function? Adenovirus-mediated gene transfer of selected proteins (secreted or intracellular) into normal islets ex vivo has demonstrated to be useful for improving islet transplant outcomes (10,11,33,34). Using this adenoviral-mediated ex vivo gene transfer approach we could start unraveling whether enhancing the PKB/Akt signaling pathway in human islet cells leads to beneficial outcomes in human islet engraftment and survival in mice.
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PKB/Akt has emerged as the focal point for many intracellular signal transduction pathways, regulating multiple cellular processes such as apoptosis, proliferation, angiogenesis and glucose metabolism (15-17). Among the myriad of downstream substrates or modulators, which could influence PKB/Akt intracellular functions, are BAD, caspase-9, glycogen synthase kinase-3 a/ß (GSK-3a/ß), the family of forkhead box (Fox) transcription factors and murine double minute 2 (MDM2). PKB/Akt has been shown to phosphorylate these proteins impeding them from performing their pro-apoptotic functions. Phosphorylation/activation of some of these proteins such as MDM2, Fox transcription factors, and others such as p27 and Cyclin D (in this case through GSK-3a/ß) have been shown to be also implicated in the proliferative effects of PKB/Akt. PKB/Akt-induced phosphorylation of the endothelial nitric oxide synthase (eNOS) at Ser1179, and consequent activation leads to NO production by the endothelial cells. Nitric oxide production by eNOS is a key regulator of vascular angiogenesis and vascular remodeling after injury. However, the exact mechanism by which PKB/Akt activates/interacts with eNOS is not known.