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Abstract

  1. Top of page
  2. Abstract
  3. APOPTOSIS IN PANCREATIC BETA-CELLS
  4. STIMULI OF APOPTOSIS IN PANCREATIC BETA-CELLS
  5. CASPASES: ACTIVATION AND PATHWAY
  6. FROM THE ACTIVATION OF CASPASES TO THE APOPTOTIC CHANGES IN THE MORPHOLOGY OF PANCREATIC BETA-CELLS
  7. STRATEGIES FOR PREVENTING APOPTOSIS OF ISLETS
  8. CONCLUSION
  9. Acknowledgements
  10. LITERATURE CITED

The homeostatic control of beta-cell mass in normal and pathological conditions is based on the balance of proliferation, differentiation, and death of the insulin-secreting cells. A considerable body of evidence, accumulated during the last decade, has emphasized the significance of the disregulation of the mechnanisms regulating the apoptosis of beta-cells in the sequence of events that lead to the development of diabetes. The identification of agents capable of interfering with this process needs to be based on a better understanding of the beta-cell specific pathways that are activated during apoptosis. The aim of this article is fivefold: (1) a review of the evidence for beta-cell apoptosis in Type I diabetes, Type II diabetes, and islet transplantation, (2) to review the common stimuli and their mechanisms in pancreatic beta-cell apoptosis, (3) to review the role of caspases and their activation pathway in beta-cell apoptosis, (4) to review the caspase cascade and morphological cellular changes in apoptotic beta-cells, and (5) to highlight the putative strategies for preventing pancreatic beta-cells from apoptosis. © 2004 Wiley-Liss, Inc.

The growth and development of the endocrine pancreas has been studied for many years. Questions concerning the regulation of the mass of insulin-producing beta-cells both in the normal growing pancreas and during the pathogenesis of diabetes have been highlighted in the past several years, especially the relationship between the loss of pancreatic beta-cells and apoptosis. Apoptosis, or programmed cell death (PCD), is an important mechanism for tissue modeling during development and adult life. Cell apoptosis plays an essential role in the removal of superfluous, infected, transformed, or damaged cells by activation of an intrinsic suicide program (Payne et al., 1995; Warner, 1997). In addition, this is also a fundamental mechanism leading to cell loss and disease. Pancreatic beta-cells are sensitive to a number of pro-apoptotic stimuli believed to be involved in the development of Types I and II diabetes (O'Brien et al., 1997; Mathis et al., 2001), as well as in the late complications of diabetes (Darby et al., 1997; Fukagawa et al., 2001).

During the last few years, there have been significant advances in our understanding of the pathogenetic mechanisms responsible for the beta-cell loss associated with diabetes and failure of islet transplantation. There is evidence of decreased beta-cell mass in both diabetes and islet transplantation mainly by initiation of the apoptosis process. Therefore, to understand the pathogenesis of diabetes and rationalize its treatment, one must first appreciate the basic events in beta-cell apoptosis.

APOPTOSIS IN PANCREATIC BETA-CELLS

  1. Top of page
  2. Abstract
  3. APOPTOSIS IN PANCREATIC BETA-CELLS
  4. STIMULI OF APOPTOSIS IN PANCREATIC BETA-CELLS
  5. CASPASES: ACTIVATION AND PATHWAY
  6. FROM THE ACTIVATION OF CASPASES TO THE APOPTOTIC CHANGES IN THE MORPHOLOGY OF PANCREATIC BETA-CELLS
  7. STRATEGIES FOR PREVENTING APOPTOSIS OF ISLETS
  8. CONCLUSION
  9. Acknowledgements
  10. LITERATURE CITED

Type I insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease that results from the destruction of insulin-secreting pancreatic islet beta-cells by autoreactive cells and their mediators, such as cytokines (Sjoholm, 1998; Kreuwel and Sherman, 2001). Several recent studies have investigated the role of cell apoptosis in the events leading to the immune-mediated loss of beta-cells either in vitro or ex vivo. Based on studies of pancreatic biopsies taken from diabetes-prone and diabetes-resistant BB/S rats, Lally et al. (2001) showed that the appearance of beta-cell apoptosis in the diabetes-prone group was detectable as early as 68 days of life (long before the onset of diabetes). This was followed by an acceleration of apoptosis rate around 85 days of age in the diabetes-prone group, which co-incided with the onset of hyperglycemia. Similarly, Augstein et al. (1998), using NOD (non-obese diabetic) mice, demonstrated that the frequency of apoptosis of islet cells correlated with the progression of beta-cell destruction and the decline of glycemic control.

Type II diabetes mellitus is characterized by an asymptomatic insulin resistance phase preceding the onset of diabetes, while hyperglycemia occurs when a relative insulin secretory deficiency is manifest (Cerasi et al., 2000; Mandrup-Poulsen, 2001). Studies from autopsies of subjects affected by Type II diabetes demonstrated that in some, although not all individuals with diabetes, there is a marked decrease in beta-cells mass compared to control normoglycemic subjects (Yagihashi, 1996). While a smaller beta-cells mass could result from either an insufficient proliferative capacity or from an excessive rate of cell death, there is evidence of other attributable factors. Several studies have demonstrated that in vitro, free fatty acids, glucose, sulfonylurea, and the islet cell hormone termed amylin can cause beta-cell apoptosis. This suggests that PCD may also be involved in the pathogenesis of Type II diabetes (Schwingshackl et al., 1998; Shimabukuro et al., 1998; Garratt et al., 1999; Federici et al., 2001). In the Psammomys obesus rat, an animal model of Type II diabetes mellitus, the progression from hyperinsulinemia (present in the prediabetic state) to insulin deficiency is characterized by a progressive loss of pancreatic beta-cells. These animals turn diabetic after only 4 days of a high calorie diet (Donath et al., 1999; Leibowitz et al., 2001). Cerasi et al. (2000) observed that the rate of beta-cells apoptosis is low in non-diabetic animals and increases 14-fold by 20 days after the onset of diabetes. Cell apoptosis has also been shown to be the mechanism leading to beta-cells loss in the Zucker diabetic fatty (ZDF) rat, another model of Type II diabetes. In ZDF rats, the rate of beta-cell proliferation or neogenesis is not reduced and hyperglycemia is detected when the expansion of beta-cells mass is no longer capable of compensating for the degree of insulin resistance and beta-cells apoptosis (Pick et al., 1998; Farilla et al., 2002).

Transplantation of islets of Langerhans represents a viable therapeutic approach for the treatment of Type I diabetes. Unfortunately, several problems can occur after allogenetic islet transplantation: primary non-function, rejection, and the recurrence of autoimmune disease, which involves attacks by the recipient's cytokines, T-cells, natural killer (NK) cells, and monocytes on the donor's beta-cells, leading to beta-cell destruction. Various research groups have conducted investigations on the mechanisms regulating death or survival of insulin-producing cells in cultured islets. Paraskevas et al. (2000) described the apoptosis of islet cells after human islet isolation. Human islets were enzymatically isolated from cadaveric donor pancreata and cultured in vitro up to 7 days. Transglutaminase (TG) activity and DNA fragmentation increased by 1,000% and 1,890%, respectively. This corresponded to the appearance of pyknotic nuclei under light microscopy, the presence of apoptotic bodies under electron microscopy, and the demonstration of terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL)-positive cells. These were present primarily in a distribution that corresponded to the insulin-immunoreactive cells. At 5 days, 31.4% of the islet cells were TUNEL positive. Using canine islets isolated with Liberase CI for up to 5 days in culture, Rosenberg et al. (1999) observed that the apoptotic index, as determined by TUNEL assay increased from 5.1% on the day of isolation to 60.2% on day 5, affecting mostly beta-cells. It has been reported recently that even in optimal conditions, approximately 60% of transplanted islet tissue was lost 3 days after syngeneic transplantation, and both apoptosis and necrosis contributed to beta-cell death. Increased apoptosis and reduced beta-cells mass were also found in islets exposed to chronic hyperglycemia, suggesting that sustained hyperglycemia increased apoptosis in transplanted beta-cells (Biarnes et al., 2002).

STIMULI OF APOPTOSIS IN PANCREATIC BETA-CELLS

  1. Top of page
  2. Abstract
  3. APOPTOSIS IN PANCREATIC BETA-CELLS
  4. STIMULI OF APOPTOSIS IN PANCREATIC BETA-CELLS
  5. CASPASES: ACTIVATION AND PATHWAY
  6. FROM THE ACTIVATION OF CASPASES TO THE APOPTOTIC CHANGES IN THE MORPHOLOGY OF PANCREATIC BETA-CELLS
  7. STRATEGIES FOR PREVENTING APOPTOSIS OF ISLETS
  8. CONCLUSION
  9. Acknowledgements
  10. LITERATURE CITED

Many factors have been identified as pro-apoptotic signals in eukaryotic cells (Payne et al., 1995). In studies using in vitro models, it has been shown that both direct cytotoxic (T-cell-mediated) and indirect cytokine-, nitric oxide (NO)- or free radical-, and Fas ligand-dependent mechanisms are responsible for beta-cell apoptosis. It has been recognized that cytokines, lipotoxity, and glucotoxity are three main stimuli for beta-cell apoptosis.

Beta-cell death and cytokines

A great body of evidence indicates that cytokines secreted by islet-infiltrating cells (CD4+ and CD8+ T-lymphocytes, B-lymphocytes, NK, and macrophages) might have an important role in the pathogenesis in Type I diabetes (Gottlieb and Hayward, 2002). In NOD mice, macrophages are the first inflammatory cells to infiltrate the islets, where they may engulf apoptotic beta-cells. A current hypothesis is that the pathogenic immune response is mediated by a Th1 subset of T-cells, whereas the protective response is mediated by a Th2 subset of T-cells. Th1 and Th2 cells produce a different cytokine pattern. Th1 cells secrete IL-2, IFN-gamma, and TGF-beta and activate cell-mediated immunity, whereas Th2 cells secrete IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 and stimulate humoral immunity.

Many studies support this hypothesis. As a matter of fact, both IL-4 and TGF-beta, when transgenically expressed in the pancreatic beta-cells of NOD mice, prevented autoimmune diabetes. In addition, macrophages produce IL-1 beta, TGF-alpha, and IFN-gamma that are referred to as pro-inflammatory cytokines. A correlation has been observed between beta-cell destructive insulitis and expression of pro-inflammatory cytokines. Several cytokines potentially involved in the pathogenesis of Type I diabetes have been found to be expressed in the insulitis lesions of NOD mice, BB rats and man. Although, this observation does not directly prove the pathogenic role of these cytokines, it has been observed that IL1-beta, TGF-alpha, and INF-gamma, when combined, induce functional and morphological damage to beta-cells in both rodent and human islets in a dose and time-dependent manner (Trincavelli et al., 2002).

The molecular events of cytokine-induced apoptosis have been investigated. Signal transduction by these cytokines involves: (i) binding to specific receptors, (ii) signal transduction by cytosolic kinases (especially the purported mitogen- and stress-activated protein kinases) and/or phosphatases, such as c-Jun NH2-terminal kinase (the interleukin-1β (IL1-β) induced phosphorylation of the p38 mitogen-activated protein kinase) and mitogen- and stress-activated protein kinase 1 (MSK1), both in rat insulin-producing RINm5F cells (Saldeen et al., 2001), (iii) mobilization of diverse transcription factors with nuclear factor kappaB (NF-kappaB), AP-1, and STAT-1 probably playing key roles for beta-cell apoptosis, (iv) upregulation or downregulation of gene transcription. IFN-gamma and tumor necrosis factor (TNF)-alpha synergistically induced MHC class II expression on insulinoma cells through the induction of CIITA (Eizirik and Mandrup-Poulsen, 2001).

The role of NO in beta-cell apoptosis has been investigated. Incubation of human islets with a combination of IL1-beta + TNF-alpha + IFN-gamma determined upregulation of the expression of inducible NO synthase (iNOS) and the subsequent synthesis of the radical NO as well as of other free radicals such as peroxynitrite (ONOO−) and superoxide (O2−) that contribute to beta-cells damage. NO impairs beta-cell function by blocking the enzyme aconitase and by inducing DNA strand breaks. Previous reports showed that NO could induce Fas expression on beta-cells so that they might be destroyed by T-lymphocytes (Sjoholm, 1998; Hugues et al., 2002).

However, at present, the role of NO in beta-cell damage is controversial because some observations suggest that it is unlikely that NO might be responsible for the massive cytokine-induced beta-cell destruction, although the increased level of NO was observed (Liu et al., 2000; Zumsteg et al., 2000). iNOS-deficient mouse islets, treated with a combination of IL1-beta+TNF-alpha+IFN-gamma, expressed Fas mRNA in response to cytokines similarly to wild-type islets and the murine NIT-1 beta-cell line. In addition, the accumulation of Fas-specific mRNA in islets following exposure to pro-inflammatory cytokines correlated directly with an increased surface expression of Fas comparably in wild-type, iNOS-deficient islets, and NIT-1. These results suggested that Fas mRNA transcription and Fas protein expression in murine islets are independent of NO production. Moreover, it has been observed that inhibition of NO synthesis, in vitro, in isolated pancreatic beta-cells as well as in vivo, produced only a partial protection from cytokine-mediated cytotoxicity. On the other hand, it has been demonstrated that prolonged incubation with IL1-beta+TNF-alpha+IFN-gamma revealed increased apoptotic cell death in wild-type islets as well as in Fas-deficient islets. Thus cytokines are able to induce PCD in beta-cells independently of any Fas-FasL mediated signaling and NO production.

The investigation of the role of ceramide in islet cell apoptosis has indicated that the activation of the sphingomielynase pathway mediates the effect of cytokines. A recent report has shown that beta-TC3 cells treated with a combination of IL1-beta + TNF-alpha + IFN-gamma did not increase ceramide accumulation even if exogenous ceramide analogs reproduced the effects of cytokines on the beta-cells. These findings suggest that the sphingomielynase/ceramide pathway is not involved in cytokine-induced beta-cell death (Major et al., 2000). These data demonstrate that the mechanism(s) by which cytokines destroy beta-cells are complex and, although under active investigation, they are far from being elucidated.

Viral infections may trigger the autoimmune assault leading to Type I diabetes mellitus. Double-stranded RNA (dsRNA) is produced by many viruses during their replicative cycle. The dsRNA, tested as synthetic poly(IC) (PIC), in synergism with the pro-inflammatory cytokines interferon-gamma (IFN-gamma) and/or IL-1 beta, results in NO production, Fas expression, beta-cell dysfunction, and death. Activation of the transcription factor NF-kappa B plays a central role in at least part of the deleterious effects of dsRNA in pancreatic beta-cells (Liu et al., 2002). This finding is supported by the study of Heitmeier et al. (2001), who showed that viral replicative intermediate dsRNA stimulates beta-cell production of pro-IL-1-beta, and following cleavage to its mature form by IFN-gamma-activated ICE, IL-1 then initiates beta-cell damage in a NO-dependent fashion.

It has been observed in animal models that cytokines, such as IL-4 and IL-10, can be protective, being able to counteract the adverse effects of pro-inflammatory cytokines and reducing beta-cell damage (Rabinovitch, 1998). In this regard, Guerra et al. (2001) have shown that: (1) isolated human islets treated in vitro with IL1-beta + TNF-alpha + INF-gamma showed a significant decrease of glucose-stimulated insulin release, (2) islets precultured with IL-4 and IL-10 followed by IFN gamma + TNFalpha + IL-1beta showed a glucose-stimulated insulin release similar to control untreated islets, (3) preincubation with IL-4 and IL-10 significantly reduced the rate of islet cell death induced by IL1-beta + TNF-alpha + IFN-gamma (Guerra et al., 2001).

Beta-cell death and lipotoxicity

Physiological plasma levels of FFA are important for beta-cell function. And fatty acid oxidation is necessary for its stimulation of insulin secretion. Long-chain acyl-CoA (LC-CoA) controls several aspects of the beta-cell function including activation of certain types of protein kinase C (PKC), modulation of ion channels, protein acylation, ceramide- and/or NO (NO)-mediated apoptosis, and binding to nuclear transcriptional factors. It has been reported that FAA overload in skeletal muscle causes insulin resistance; in myocardium, it impaired cardiac function; and in pancreatic islets, it caused beta-cell dysfunction, apoptosis, and diabetes (Unger and Zhou, 2001).

In order to better understand the phenomenon of lipotoxicity in human beta-cells, Lupi et al. (2002) evaluated the effects of FFA on the function and survival of isolated human islets and investigated some of the possible mechanisms. Insulin content and glucose-stimulated insulin release and glucose utilization and oxidation were significantly reduced in islets precultured with FFA compared to control islets. In addition, it was observed that exposure to FFA induced a significant increase of the amount of apoptotic beta-cells. FFA-induced islet cell death was blocked by inhibition of upstream caspases, partially prevented by inhibiton of ceramide synthesis or serine proteases activity, whereas inhibition of NO synthesis had no effect. A marked decrease of bcl-2 mRNA expression in the islets cultured with FFA was detected as well. Thus, prolonged exposure to FFA has cytostatic and pro-apoptotic effects on human pancreatic beta-cells. The cytostatic action is likely to be due to the FFA-induced reduction of intraislet glucose metabolism and pro-apoptotic effects, which are mainly caspase-mediated, partially dependent on ceramide pathway, and possibly Bcl-2 regulated.

Beta-cell death and glucose toxicity

Glucose can regulate gene expression of islets and furthermore change cell status under physiological or pathophysiological conditions. In islets from hyperglycemic rats, 4 weeks after partial pancreatectomy (Px), a variety of protective genes were upregulated with markedly increased expression of the anti-oxidant genes heme oxygenase-1 and glutathione peroxidase, and the anti-apoptotic gene A20. Cu/Zn-superoxide dismutase (SOD) and Mn-SOD were modestly induced, nuclear factor-kappaB was activated and Bcl-2 was modestly reduced; however, several other stress genes (catalase, heat-shock protein 70, and p53) were unaltered (Laybutt et al., 2002). The expression of the early response gene c-Myc in rat pancreatic beta-cells has been found to be stimulated by high glucose in a Ca2+-dependent manner and by cAMP. These effects of high glucose were reproduced by high potassium-induced depolarization or dibutyryl-cAMP and were inhibited by agents decreasing cytosolic Ca2+or cAMP concentrations (Jonas et al., 2001).

In vitro exposure of islets from non-diabetic organ donors to high glucose levels resulted in increased production and release of IL-1beta, followed by NF-kappaB activation, Fas upregulation, caspases-8 and -3 activation, decreased FADD-like IL1β-converting enzyme (FLICE)-inhibitory proteins (FLIP) expression, DNA fragmentation, and impaired beta-cells function (Maedler et al., 2002a,b). In Type II diabetes, chronic hyperglycemia is suggested to be detrimental to pancreatic beta-cells, causing impaired insulin secretion. Several studies have recently investigated the effect of glucose toxicity on beta-cell survival. All together, these studies indicate that glucose toxicity might play a role in facilitating human beta-cell apoptosis as a consequence of increased expression of pro-apoptotic signals, belonging both to the extrinsic (FasL) and intrinsic (Bad, Bid) pathways (Federici et al., 2001; Maedler et al., 2001). Some, but not all, converge to alter mitochondrial membrane potential. One crucial point to balance the role of glucotoxicity with respect to (cell apoptosis concerns which glucose metabolic pathways provoke cell suicide. Hexosamine metabolism is a strong candidate due to the peculiar sensitivity of beta-cells to this pathway of glucose metabolism. However, it has also been proposed that non-enzymatic glycosylation with the synthesis of AGE products could have a role (Yoshii et al., 2002).

Several other factors have also been found to play a minor role in apoptosis of pancreatic beta-cells. Human amylin (HA) induces apoptosis through stimulation of expression and activation of c-Jun. Co-expression and dimerization of c-Jun and c-fos or ATF-2 may be important for activation of the downstream apoptotic process (Zhang et al., 2002).

CASPASES: ACTIVATION AND PATHWAY

  1. Top of page
  2. Abstract
  3. APOPTOSIS IN PANCREATIC BETA-CELLS
  4. STIMULI OF APOPTOSIS IN PANCREATIC BETA-CELLS
  5. CASPASES: ACTIVATION AND PATHWAY
  6. FROM THE ACTIVATION OF CASPASES TO THE APOPTOTIC CHANGES IN THE MORPHOLOGY OF PANCREATIC BETA-CELLS
  7. STRATEGIES FOR PREVENTING APOPTOSIS OF ISLETS
  8. CONCLUSION
  9. Acknowledgements
  10. LITERATURE CITED

In the past decade, several genes regulating cell apoptosis have been identified. Those genes are often termed “family” or “superfamily” to describe the degree of similarity of action or biochemical structure of the individual components. An example is represented by the Bcl-2 family (Chao and Korsmeyer, 1998), the TNF superfamily (Gupta, 2001), the TNF receptor superfamily (Idriss and Naismith, 2000), the apoptotic protease activating factor 1 (Apaf-1), C. elegans death-4 defective gene (CED-4) proteins (Cecconi, 1999). At least four apoptotic pathways have been shown to be capable of inducing beta-cell death. The complexity of this system is further underscored by the participation of many independent proteinases, termed caspases, that are part of these apoptotic pathways and are key players in controlling the events leading to cell apoptosis (Fig. 1). Interestingly, many of them need cysteine residue to exert their activity.

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Figure 1. The pathway of activation of the caspase cascade. A variety of stimuli including chemicals, physical insults, cytokines, as well as the withdrawal of trophic factors can serve as initiators of the cell death pathway. These stimuli promote the release of caspase activators from mitochondria and result in the activation of “initiator” caspases, which then lead to the cleavage and activation of “effector” caspases. The effector caspase interact with a variety of specific cellular proteins, thus accounting directly or indirectly for morphological and biochemical characteristics of cells undergoing apoptosis. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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The term caspase was chosen to denote the cysteine requiring ASPartate proteASE activity of these enzymes. These cysteine-rich proteases are key regulators of cell apoptosis. To date, 14 distinct caspases (caspases-1–14) have been identified. Phylogenetic analysis of their sequence characteristics showed that the human caspase sequences cluster into two subfamilies: they are termed ICE (caspase-1) and the CED-3 (C. elegans caspase) subfamily, based on the characteristics of the full-length pro-enzymes. Based on the sequence similarity in their protease domains, caspases are further divided into three groups. The first group consists of inflammatory caspases and includes caspases-1, -4, -5, -11, -12, -13, and -14. The second group contains caspases-2 and -9. The rest of the caspases form the third group (Cohen, 1997; Thornberry, 1997; Wang and Gu, 2001; Lamkanfi et al., 2002). An additional classification of caspases distinguishes initiator versus effector caspases, depending on whether the activity is that of an early activator (initiator) or late mediator (effector) on the processes leading to apoptosis. This latter classification is used hereafter for the description of the individual member proteins.

Activation of caspases

As described above, all caspases are synthesized as inactive pro-enzymes and each pro-caspase contains a NH2-terminal peptide terminus of a variable length (pro-domains) (Fig. 2). In most cases, there are over 100 amino acids in a pro-caspase, while generally less than 30 amino acids are present in the biologically active caspases. Such long pro-domains contain distinct motifs with important regulatory functions in the events that lead an individual caspase to function as part of a complex network of enzymes that, when sequentially activated, leads to cell death. These regions cleaved from the mature enzyme include the following domains: death effector domain (DED), caspase recruitment domain (CARD), and the death-inducing domain (DID). These amino acid sequences, similar to the so-called DD domain (death domain), which are characteristically present in cell death adapter proteins, consist of six alpha-helices and have a similar overall folding structure. These domains mediate the homophilic interaction between pro-caspases and their adapters and play an important role in pro-caspase activation. While charge–charge interactions govern CARD–CARD and DD–DD association, hydrophobic interactions govern DED–DED interaction. The short pro-domains of executioner caspases seem to inhibit caspase activation, perhaps operating as a self-limiting mechanism to slow down or diminish cell apoptosis (Budihardjo et al., 1999; Martin, 2001; Weber and Vincenz, 2001; Gozani et al., 2002). All caspases contain a conserved QACXG pentapeptide active-site motif (where X is either R, Q, or G). During the process that leads to the production of mature caspases, the pro-domain and the linker peptide are cleaved at specific Asp residues (Table 1) (Cohen, 1997).

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Figure 2. Caspase structure and activation. The pro-caspase is inactive and its typical structure contains three parts: a pro-domain, a large subunit, and a small subunit. The length of pro-domain vary among caspases and some of that has death effector domain feature (DED) or caspse recriutment domain feature (CARD), pro-enzyme is cleaved at caspase cleavage sequences (Aps-X), two large and two small subunits combine to form the active tetrameric enzyme. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Table 1. Alternative names and active-sites of human caspases
CaspaseAlternative nameActive sequenceCleavage site
Caspase-1ICEQACRGAsp-103, Asp119, Asp-297, Asp-316
Caspase-2Nedd2, ICH-1QACRGAsp-152 (exact site not known), Asp-316, Asp-330 (cleavage sites are based on equivalent sites in caspase-2)
Caspase-3CPP32, Yama, apopainQACRGAsp-9/Asp-28, Asp-175
Caspase-4ICErelII, TX, ICH-2QACRGAsp-104, Asp-270, Asp-289
Caspase-5ICErelIII, TYQACRGAsp-121 (exact site not known), Asp-311, Asp-330
Caspase-6Mch2QACRGAsp-23, Asp-179, Asp-193
Caspase-7Mch3, ICE-LAP3, CMH-1QACRGAsp-23, Asp-198
Caspase-8MACH, FLICE, Mch5QACQGAsp-210, Asp-216, Asp-374, Asp-384
Caspase-9ICE-LAP6, Mch6QACGGAsp-130 (exact site not known), Asp-315 (cleavage by granzyme B)/Asp-330 (cleavage by caspase-3)
Caspase-10Mch4QACQGAsp-219, Asp-372

Initiator and activator caspases utilize different mechanisms to generate the production of the active protein. Protein oligomerization is the recognized common mechanism of activation of all initiator caspases. Apoptotic signals trigger oligomerization of death adapter proteins (e.g., CED-4, Apaf-1, and FADD); death adapter oligomers, in turn, oligomerize pro-caspases, leading to their autoproteolytic activation. Active initiator caspases then process and activate effector pro-caspases. Finally, effector caspases cleave various death substrates to induce cell death (Yang et al., 1998a; Salvesen and Dixit, 1999; Chen and Wang, 2002).

Effector pro-caspases are activated by either initiator caspases or other proteases (transactivation). In vitro studies have shown that pro-caspases-3 and -7 can be activated by caspases-6, -8, -9, and -10, however, the extent to which this transactivation occurs in vivo is not yet fully understood. Effector caspases, as well as some initiator caspases, can also be activated in vitro by granzyme B. The transactivation appears to be a two-step process, which has been well characterized for the activation of caspase-3 by granzyme B. The first proteolytic cleavage of caspase-3 carried out by granzyme B occurs in the linker region between the large and small subunits. This leads to the generation of a partially active intermediate that may resemble the mature caspase in structure. In the second step, the partially active intermediate processes itself to generate the fully active, mature caspase. This self-cleavage produces the short inhibitory prodomain from the large subunit (Talanian et al., 1997a; Stennicke et al., 1998; Yang et al., 1998a; Slee et al., 1999b).

The cleavage specificity is largely defined by a tetrapeptide recognition sites in which enzymatic cleavage occurs on the carboxyl side of an Asp residue (P1). The P4 cleavage site residue (where the numbering runs from the C towards the N direction) is the most important residue in determining the substrate specificity. Group I enzymes (human caspases-1, -4, and -5) prefer bulky hydrophobic or aromatic residues such as Tyr, Trp or Leu. Group II caspases (human caspases-2, -3, and -7) prefer a P4 Asp and a P3 Glu, while Group III caspases (caspases-6, -8, -9, -10) prefer aliphatic P4 residues such as Ile, Leu, or Val. There is substantial but not complete overlap between this system of classification and that based on sequence homology or shared function. Group I enzymes are also part of the ICE (caspase-1) homology subfamily and function primarily in inflammatory processes that lead to cell apoptosis. Groups II and III caspases together constitute the CED-3 homology subfamily. Most of the Group III caspases (caspases-8, -9, and -10) are characterized by long pro-domain enzymes with initiator functions in apoptosis, while caspases-3 and -7, which are part of Group II, are downstream effector caspases, and they posses short pro-domains. Caspases-2 and -6 may represent exceptions to this pattern. Group III member caspase-6 has a short pro-domain and, as evidenced by its cleavage of nuclear laminas, appears to function as an effector caspase. Caspase-2, although a member of Group II, has a long N-terminal pro-domain with a CARD-like sequence (similar feature in caspases-9 and -8), and can be recruited to ligated death-receptor complexes via interaction with adaptor proteins (Stennicke and Salvesen, 1997; Talanian et al., 1997b; Chou et al., 1998; Slee et al., 1999a; Chang and Yang, 2000; Rotonda et al., 2001; Schickling et al., 2001).

After having been cleaved, the large subunit and the small subunit (termed homodimer p20 and p10) are arranged in twofold rotational symmetry and interact in a heterodimer. The association of two heterodimers forms the pro-teolytic tetramer with the two adjacent small subunits surrounded by two large subunits. Each p20-p10 heterodimer forms a single globular domain and the core of the globular domain is a six-stranded beta-sheet structure, flanked on either side by alpha-helices. These two heterodimers associate with each other primarily through the interaction between the p10 subunits. Each caspase tetramer has two cavity-shaped active sites formed by amino acids from both the p20 and p10 subunits and these two active sites are likely to function independently (Walker et al., 1994; Talanian et al., 1996; Kumar, 1999; Zimmermann et al., 2001).

The subunit, tissue distribution, substrates and function of caspase are summarized in Table 2.

Table 2. Substrates and function of caspases
CaspaseSize and subunitsTissue distributionSubstratesFunction (and notes)
Caspase-1Pro-enzyme: 45,158 Da; active: p20 and p10Spleen, lung, liver, heart, skeletal muscle, kidney, and testisPreinterleukin-1β; interleukin-18, laminsProcessing of interleukins (inflammation); can also induce apoptosis depending on isoform and if overexpressed
Caspase-2Pro-enzyme: 48,855 Da; active: p12/14 and p19Central nervous system, liver, kidney, and lung during embryonic developmentGolgin-160, laminsApoptosis
Caspase-3Pro-enzyme: 31,594 Da; active: p17 and p12Widely distributed, with high expression in cell lines of lymphocytic originPARP, SREBs, gelsolin, caspases-6, -7, -9, DNA-PK, MDM2, Gas2, fodrin, b-catenin, lamins, NuMA, HnRNP proteins, topoisomerase I, FAK, calpastatin, p21Waf1, presenelin2, ICADApoptosis
Caspase-4Pro-enzyme: 43,262 Da; active: p20 and p10Lung, liver, ovary, and placenta, barely detected in brain.Caspase-1Inflammation/apoptosis
Caspase-5Pro-enzyme: 47,814 Da; active: p20 and p10Lung, liver, and skeletal muscleMaxInflammation/apoptosis
Caspase-6Pro-enzyme: 33,409 Da; active: p18 and p11Lung, liver, and skeletal musclePARP, lamins, NuMA, FAK, caspase-3, keratin-18Apoptosis
Caspase-7Pro-enzyme: 34,276 Da; active: p20 and p11Lung, skeletal muscle, liver, kidney, spleen, heart, and testis; no expression in the brainPARP, Gas2, SREB1, EMAP II, FAK, calpastatin, p21Waf1Apoptosis
Caspase-8Pro-enzyme: 55,391 Da; active: p18 and p10Highest expression in peripheral blood leukocytes, spleen, thymus, and liver; barely detectable in brain, testis, and skeletal muscleCaspases-3, -4, -6, -7, -9, -10, -13, PARP BidApoptosis (death receptors)
Caspase-9Pro-enzyme: 46,322 Da; active: p35 and p10Highly expressed in the heart, moderate expression in liver, skeletal muscle, and pancreas; low levels in all other tissuesCaspase-3, pro-caspase-9, caspase-7, PARPApoptosis
Caspase-10Pro-enzyme: 58,878 Da; active: p23/17 and p12Detectable in most tissues; lowest expression is seen in brain, kidney, prostate, testis, and colonCaspases-3, -4, -6, -7, -8, -9Apoptosis (death receptors)
Caspase-11Pro-enzyme: 43,262 Da; active: p20 and p10Brain microgliaCaspases-3, -1Involved in inflammation and apoptosis
Caspase-12Pro-enzyme: 48,325 Da; active: p38 and p28Predominantly in the endoplasmic reticulumCaspases-1, -4, -5, -11Involved in mediating apoptosis following ER stress
Caspase-13Proenzyme: 46,371 Da; active p35 and p10Peripheral blood lymphocytes, spleen, and placentaGranzyme B, caspase-8Involved in inflammation
Caspase-14Pro-enzyme: 27,679 Da; active: p18 and p11Epidermal cellsCaspases-8, -10, granzyme BInvolved in inflammation

Pathways of caspase activation in apoptosis

Caspases work coordinately and sequentially in a process leading to cell apoptosis (Nunez et al., 1998). Initiator caspases (e.g., caspases-1, -8, -9, -10) are the upstream activators of the effector caspases (e.g., caspases-2, -3, -6, -7) (Kaplowitz, 2000). Effector caspases are the apoptosis executioners and their activities (Seol et al., 2001) lead to the characteristic apoptotic morphological changes such as membrane blebbing, cytoplasmic and nuclear condensation, DNA fragmentation, and formation of apoptotic bodies.

FAS pathway

CD95 (also termed Fas or APO-1) belongs to the TNF-receptor family of proteins and is expressed on most cell types (Stanger, 1996; Sharma et al., 2000). Upon binding of its ligand (FasL), CD95 oligomers are formed and a signal to enter the apoptotic route is transmitted to cells via a 70 amino acid long cytoplasmic domain (termed DD), which is highly conserved between CD95 and TNF receptor 1.

In NOD mice, islet expression of the Fas was increased in islets from female mice 15 weeks of age as compared to corresponding males, but islet expression of FasL could not be detected (Ingelsson et al., 1998). Apoptosis via Fas/Fas ligand (FasL) interactions has been proposed to be a major T-cell-mediated effector mechanism in autoimmune diabetes. Loweth et al. (1998) showed that human islets of Langerhans express Fas ligand and undergo apoptosis in response to IL1β and Fas ligation. It is notable that there were some contrary reportings for the role of Fas-FasL in apoptosis of beta-cells. By administering anti-Fas ligand (FasL) Ab and by grafting Fas-deficient neonatal pancreas from NOD-lpr/lpr mice, Kim et al. (1999) showed that Fas-FasL interaction is not involved in the development of diabetes in NOD mice. In addition, few Fas-positive cells were isolated immediately before the development of diabetes (Thomas et al., 1999).

One of the possible explanations for this discrepancy is the inducibility of Fas expression in beta-cells by cytokines and high glucose. Inflammatory insults specifically induce translocation of Fas to the beta-cell surface (Augstein et al., 2002) and increased glucose concentrations induce Fas expression and beta-cells apoptosis.

It remains controversial whether beta-cells truly express FasL. This is largely because FasL expression has only been demonstrable by immunohistochemistry and not by flow cytometry. The following experiments supplied indirect evidence for the involvement of FasL in beta-cell apoptosis. Beta-cells from FasL transgenic NOD mice are more susceptible to cytokine-induced apoptosis than wild-type beta-cells. This is consistent with the hypothesis that proposes if beta-cells express FasL, then the interaction between Fas and FasL would be able to mediate cell death even in the absence of T-cells (Petrovsky et al., 2002). In addition, the administration of anti-FasL antibody at 2–4 weeks of age to NOD mice prevented insulitis and diabetes. This suggests that Fas/FasL interactions contribute to CD4(+) T-cell-mediated beta-cell destruction and play an essential role in the initiation of autoimmune diabetes (Nakayama et al., 2002).

In the advanced stages of Type II diabetes, Fas-to-Fas-ligand (FasL) interaction is also involved in the decline in beta-cell mass. Maedler et al., 2001, 2002b observed upregulation of Fas in beta-cells of Type II diabetic patients relative to non-diabetic control subjects. In vitro exposure of islets from non-diabetic organ donors to high glucose levels induced Fas expression, caspases-8 and -3 activation, and beta-cell apoptosis. Upregulation of the Fas receptor by elevated glucose levels may contribute to beta-cell destruction by the constitutively expressed FasL independent of an autoimmune reaction, thus providing a link between Types I and II diabetes.

To date, three different pathways have been described by which CD95 can induce apoptosis. The first pathway requires the activation of caspase-8. In the second pathway, the accessory protein DAXX, which has no evident death domain, binds to the CD95-death domain by heterotypic interaction with other (non-caspase) proteins (Yang et al., 1997). In this cascade of protein activation, c-Jun NH2-terminal kinase (JNK) pathway of apoptosis is activated. This cascade can be blocked by Bcl-2 but not by FLIP (Hofmann et al., 2001). In the third pathway, the activation of caspase-2 leads to the activation of the FAS-dependent pathway, and two other accessory molecules referred to as receptor interacting protein (RIP) and RIP-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD) bind to CD95 by homotypic interactions between their death domains. This complex recruits pro-enzymatic caspase-2, which thereby is converted into its active form and then triggers the caspase cascade (Grimm et al., 1996; Scaffidi et al., 1998; Villunger et al., 2000). Most caspases in the upstream of the cascade responsible for the Fas-mediated and TNF receptor 1 (TNFR-1)-induced cell death act by binding to the adaptor molecule FADD (Fas-Associating protein with Death Domain) and they are recruited by either one of these two receptors (Kischkel et al., 2000). The resulting aggregate, which is called death-inducing signaling complex (DISC), performs caspase-8 proteolytic activation. The active dimeric enzyme is then released from the DISC and is free to activate downstream apoptotic proteases (Gomez-Angelats and Cidlowski, 2001; Xiao et al., 2002).

Signaling by TNF family members

TNF protein family members (which include TNF; lymphotoxin (LT); CD40L; CD27L; FasL; 4-1BBL; OX40L) play an important role in various physiological and pathological processes including cell proliferation, differentiation, apoptosis, as well as modulation of immune response and induction of inflammation (Idriss and Naismith, 2000). The molecular mechanisms of signaling by three members of the TNF family, including TNF, TNF-related apoptosis-inducing ligand (TRAIL), and TNF- and ApoL-related leukocyte-expressed ligand (TALL-1), have been recently elucidated.

Signaling by TNF

TNF elicits a broad range of biological effects through two receptors, TNFR-1 and TNFR-2. TNFR-1 is expressed by most tissues and is the major signaling receptor for TNF (MacEwan, 2002). On the other hand, TNFR-2 is mostly expressed in immune cells and mediates more limited biological responses (Wajant and Scheurich, 2001).

TNFR-1, TNFR-1-associated death domain protein (TRADD), Fas receptor-associated intracellular protein with death domain (FADD), and FLICE were expressed in the pancreatic beta-cells line, MIN6 cells. Ishizuka et al. (1999) demonstrated TNF-alpha induced time- and dose-dependent apoptotic nuclear changes in these beta-cells and further suggested that TNF-alpha can cause apoptosis in pancreatic beta-cells through TNFR1-linked apoptotic factors, TRADD, FADD, and FLICE, and TNF-induced ceramide production may be involved in the pathways.

TNFR-1 can induce two distinct biological effects: apoptosis and nuclear factor-kappa B (NF-kB) activation. TNFR-1-mediated apoptosis and NF-kB activation pathways bifurcate at the level of TRADD (Hsu et al., 1996). TRADD is an important mediator of FAS and TNFR-1 associated apoptotic signaling. It contains a death domain homologous region (DDH) through which it binds to both FAS and TNFR-1. Overexpression of TRADD causes NFkB activation and apoptosis in absence of TNF. TRADD interacts with FADD to induce apoptosis (Chinnaiyan et al., 1995), while its activation of NF-kB require the interaction with the molecules TNF receptor (TNFR)-associated factor 2A (TRAF-2) and receptor-interacting protein (RIP) (Yu et al., 1999). FADD is a cytoplasmic protein that interacts with FAS to initiate apoptosis. DED of FADD binds to an analogous DED domain present in tandem in the proform of the caspase-8 protein. Recruitment of caspase-8 to the Fas receptor results in oligomerization of the caspase-8 protein, which in turn drives its autoactivation through “self-cleavage.” Activated caspase-8 then activates other downstream caspases including caspase-9, thereby commiting the cell to undergo apoptosis (Chen et al., 2001). Casper (c-FLIP, CASH, and I-CASPASE-8) interacts with FADD, caspases-8, -3, and TRAF2 through distinct domains and functions downstream of FADD and is involved in the TNFR-1- and the Fas-induced apoptosis pathway when overexpressed in mammalian cells (Shu et al., 1997). The apoptosis signaling that is induced by the FADD-Casper-caspase-8 cascade is under a negative feedback mechanism through its intrinsic ability to activate the anti-apoptotic transcription factor NF-kB (Hu et al., 2000).

Signaling by TRAIL

TRAIL is a member of the TNF family and is constitutively expressed in most normal human tissues (Gochuico et al., 2000). TRAIL-mediated apoptosis is induced via two receptors, TRAIL-R1/DR4 and TRAIL-R2/DR5, which can also activate NF-kB.

The majority of the human beta-cells express TRAIL receptors-R1, -R2, -R3, -R4 and/or TRAIL. TRAIL induced much stronger cytotoxicity and apoptosis to beta-cell lines CM and HP62 than did FasL, TNF-alpha, LTalpha1beta2, LTalpha2beta1, LIGHT, and IFN-gamma. The cytotoxicity and apoptosis induced by TRAIL to beta-cell lines CM were inhibited competitively by soluble TRAIL receptors, R1, R2, R3, or R4. Treatment of these beta-cells with antibodies against TRAIL receptors was able to block the cytotoxicity of TRAIL to these cells. Beta-cell antigen-specific cytotoxic T-lymphocyte (CTL) (CD4(+) and CD8(+)) clones express TRAIL, suggesting that these cells are potential sources of TRAIL-inducing beta-cell destruction (Ou et al., 2002). Thomas et al. (2001) also demonstrated that three major mechanisms are involved in apoptosis of freshly isolated islet cells(II-APO). These mechanisms are: caspase-6 activation, a TRAIL-induced apoptosis pathway, and the mitochondrial-associated apoptosis pathway.

Both TRAIL-R1 and TRAIL-R2 contain a “death domain” that is required for TRAIL-R1- and TRAIL-R2-induced apoptosis (Schneider et al., 1997; Walczak et al., 1997). Two additional receptors, TRAIL-R3/DcR1 and TRAIL-R4, are also capable of binding to TRAIL. TRAIL-R3 does not have a cytoplasmic domain and can protect cells against TRAIL-induced apoptosis when overexpressed in eukariotic cells. The signaling capacity of TRAIL-R4 is similar to that of TRAIL-R1 and TRAIL-R2 with respect to NF-kB activation, but differs in its inability to induce apoptosis. Yet TRAIL-R4 retains a C-terminal element containing one-third of a consensus death domain motif. Overexpression of TRAIL-R4 protects cells against TRAIL-induced apoptosis and activates the transcription factor NF-kB. Thus, TRAIL-R3 and TRAIL-R4 may both be protective receptors, either by acting as “decoy” receptors or through transducing an anti-apoptotic signal. TRAIL receptors induce apoptosis, NF-kB and JNK activation via distinct signaling pathways. Interestingly, the activation of NF-kB is not sufficient for protecting cells from TRAIL-induced apoptosis (Hu et al., 1999). The proteins that transduce the apoptosis signal from TRAIL-R1 and TRAIL-R2 to downstream caspases are not known.

It is notable that normal primary islet cells from most donors are resistant to the cytotoxicity mediated by TRAIL. However, treatment with an inhibitor of protein synthesis (cycloheximide) or with an enzyme (PI-PLC) that can remove TRAIL-R3 from the islet-cell membrane was able to increase the susceptibility of TRAIL-resistant primary islet cells to the TRAIL death pathway. That means unidentified inhibitors of the TRAIL death pathway are present in normal islet cells (Ou et al., 2002).

Granzyme-B

Granzymes are neutral serine proteases which are stored in specialized lytic granules of CTLs and in NK cells. A number of granzymes (A–G) have been isolated and cloned from mouse CTLs and NK cells. Among those, granzyme B has been shown to be involved in target cell apoptosis during lymphocyte-mediated cytotoxicity. Exocytosis of granzyme-containing granules in the cytoplasm of the target cell leads to the induction of DNA fragmentation and apoptosis (Greenberg, 1996). A mutation introduced into the granzyme B locus leads to a severe defect in the ability of cytotoxic lymphocytes to induce apoptosis in susceptible target cells, and reduces the severity of class I-dependent acute graft-versus-host disease (GvHD) (Pham and Ley, 1997).

Granzyme B is internalized via receptor mediated endocytosis through the mannose 6-phosphate receptor. In the presence of perforin, it directly activates caspases-3, -7, -8, -10, and induces rapid apoptotic death of the target cells (Fig. 3). Mitochondrial cytochrome-c release is the primary mechanism involved in granzyme B-induced apoptosis. The anti-apoptotic protein Bcl-2 blocks the rapid destruction of targets by granzyme B. However, granzyme B can also bypass this mitochondrial route and still promote apoptosis. The latter is a less efficient process, which requires significantly longer time to progress to the stage of phosphatidylserine externalization and to the morphological cellular changes that are characteristically associated with apoptosis (Barry et al., 2000; Motyka et al., 2000). The mitochondrial protein Smac/DIABLO has also been proposed to be involved in the mitochondrial killing pathway used by granzyme B (Pinkoski et al., 2001). Granzyme A-positive cells are found both in and surrounding the islets, implying induction prior to islet infiltration (Held et al., 1990).

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Figure 3. The role of granzyme B in apoptosis. Granzyme B is a neutral serine protease that interact with cellular caspase to induce pancreatic beta-cells apoptosis. Cytotoxic T-lymphocytes (CTLs) and natural killer (NK) cells inject apoptosis-inducing proteases, including granzyme B, into target cells through perforin channels, that can directly activate caspases-3, -7, -8, and -10. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Cytochrome-C

Recent studies provide strong evidence for an important role of mitochondria in the propagation of death signals and the final activation of the executing caspase cascade (Wakabayashi and Karbowski, 2001). Several different apoptosis-inducing stimuli (including dexamethasone, irradiation, etopoxide, anti-Fas mAb, TNF-alpha, staurosporine, ceramide, etc.) have been shown to result in mitochondrial permeability transition (PT). This has been shown to mediate the reduction of the mitochondrial transmembrane potential (MTP).

Treatment of HIT-15 cells (transformed pancreatic beta-cells) with 3 μg/ml mycophenolic acid (MPA) induced apoptosis. It was accompanied by a marked increase of caspase-2 activity (+343%) and moderate activation of caspase-9 (+150%) and caspase-3 (+145%). Release of the mitochondrial protein cytochrome-c into cytosol was also observed at a late stage (Huo et al., 2002). Simvastatin treated islets showed a decrease in Bad phosphorylation, cytochrome release, caspase-9 activation, and an increase in islet viability.

Mitochondria undergoing PT and MTP release mitochondrial proteins (e.g., cytochrome-c and apoptosis-inducing factor (AIF) into the cytoplasm (Kristal and Brown, 1999; Torres-Roca et al., 2000). Cytochrome-c can then bind to Apaf-1 (a mammalian protein with homology to Ced-4) which, in the presence of dATP, is capable of activating pro-caspase-9. Activation of caspase-9 may, in turn, lead to the activation of caspase-3 and result in cell death (Perkins et al., 2000).

Bcl-2 family

Bcl-2 protein family members are mainly localized at the outer mitochondrial membrane and they include both pro-survival and pro-apoptotic proteins. The better characterized pro-survival members of the Bcl-2 class includes Bcl-2 itself and Bcl-XL, while pro-apoptotic proteins include Bax, Bad, and Bid (Hanke, 2000; Harris and Thompson, 2000; Federici et al., 2001). Bcl-2 and Bcl-XL have three distinct domains. The BH1 domain (ligand domain), BH2 domain (poreformation domain), and the BH3 domain (membrane insertion domain). An additional domain, termed BH4, has been detected exclusively in Bcl-2 and has been found to allow for the interaction of Bcl-2 with regulatory proteins such as Raf-1, Bad, and Ced4. Bid is constituted only of the BH3 domain, while both Bad and Bid lack the transmembrane domain (Fig. 4).

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Figure 4. Schematic of the structure of the Bcl-2 proteins. The Bcl-2 family of proteins consists of pro-apoptotic (Bax, Bid, and Bak, etc.) and anti-apoptotic proteins (Bcl-2 and Bcl-XL, etc.). C-terminus TM domain is the member docking part and the cytoplasmic face of mitochondria OM, nuclear envelope and ER. BH1 is the ligand binding domain; BH2 is the poreformation domain; BH3 is the membrane insertion domain; BH4 domain allows the interaction of Blc-2 with death regulatory proteins such as Raf-1, Bad, and Ced 4.

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Bcl-2 is believed to suppress apoptosis via inhibition of the mitochondrial PT (Shimizu et al., 1998), However, in Fas-mediated apoptosis, the caspase-1-dependent mitochondrial PT and AIF release is not prevented by Bcl-2, while ceramide-induced or t-BHP-induced PT and AIF release are blocked by Bcl-2 (Susin et al., 1997).

Bax is present in the cytosol of live cells. After an appropriate death signal, Bax undergoes a conformational change and moves to the mitochondrial membrane where it causes release of mitochondrial cytochrome-c into the cytosol (Goping et al., 1998). Bid is also present in the cytosol of live cells. After Bid is cleaved and activated by caspase-8, it moves to the mitochondria where it causes release of cytochrome-c possibly by altering the conformation of Bax (Roucou et al., 2002). Similarly, Bak appears to undergo a conformational change that converts it from an inactive to an active state and promotes cytocrome-c release from mitochondria (Degenhardt et al., 2002). The viability of many cells depends on a constant or intermittent supply of cytokines or growth factors. In the absence of an apoptosis-suppressing cytokine, cells may undergo apoptosis. Bad is sequestered in the cytosol when some cytokines are present. Cytokine binding with receptors can activate phosphatidylinositol 3 (PI3) kinase, which phosphorylates Akt/protein kinase B (PKB), which in turn phosphorylates Bad. Phosphorylated Bad is sequestered in the cytosol by the 14-3-3 protein (Masters and Fu, 2001). Removal of the cytokine turns the kinase pathway off, while the phosphorylation state of Bad shifts to the dephosphorylated form. The dephosphorylated Bad then causes the release of cytochrome-c from the mitochondria. The balance between the anti-apoptotic and pro-apoptotic Bcl-2 family members may be critical to determining if a cell undergoes apoptosis.

Federici et al. (2001) compared the effect of a 5-day culture in high glucose concentration (16.7 mmol/l) versus normal glucose levels (5.5 mmol/l) or hyperosmolar control (mannitol 11 mmol/l plus glucose 5 mmol/l) on the survival of human pancreatic islets. The anti-apoptotic gene Bcl-2 was unaffected by glucose change, whereas Bcl-xl was reduced upon treatment with HG5. On the other hand, proapoptotic genes Bad, Bid, and Bik were overexpressed in the islets maintained in HG5.

Inhibitors of apoptosis

Cell apoptosis and the progression through the process leading to cell death are also regulated by a group of proteins with anti-apoptotic activity (Yang and Li, 2000). It has been demonstrated that the p35 protein obtained from baculovirus and cytokine response modifier A (CrmA) isolated from the cowpox virus are capable of inhibiting the enzymatic activity of caspases. These proteins are part of a class of agents termed inhibitors of apoptosis (IAPs). The baculovirus Cp-IAP and Op-IAP were the first members of this family to be identified based on their functional ability to complement the cell death inhibitor, p35, in mutant viruses. Five human IAP homologs have been identified. They include the neuronal apoptosis inhibitory protein (NAIP), cIAP1(CARD-containing IAP family), cIAP2, X-linked inhibitor of apoptosis protein (XIAP), and survivin (Fig. 6). These proteins contain a 70 amino acid motif termed BIR (baculovirus IAP repeat) domain near their amino-terminus. The BIR motif is present in one to three copies, and it may be necessary and sufficient for the anti-apoptotic effect of IAPs (Takahashi et al., 1998). The BIR domain can bind some caspases. Many members of the IAP family of proteins block the proteolytic activation of caspases-3 and -7 (Takahashi et al., 1998). XIAP, cIAP1, and cIAP2 have been shown to capable of preventing the proteolytic processing of pro-caspases-3, -6 and -7. This process requires blocking of the cytochrome-c induced activation of pro-caspase-9 by binding directly to pro-caspase-9. Furthermore, while they are not capable of preventing the caspase-8 induced activation of pro-caspase-3, they have been shown to inhibit the processing of caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases (Deveraux et al., 1998). Survivin and XIAP have been shown to inhibit Bax- and Fas-induced apoptosis and both were demonstrated to inhibit pro-caspase-7 processing. It has been shown that, in vitro, survivin and XIAP are capable of binding to active caspases-3 and -7, but not to their unactivated proforms (Ambrosini et al., 1997). This information supports the notion that survivin and XIAP act at the level of the executioner caspases-3 and -7 and do not act at the level of initiator caspases, as indicated by their inability of interacting with caspase-8 (Fig. 5). In a cell-free system, survivin has been shown to inhibit cytochrome-C dependent caspase-activity and, in vivo, to be capable of preventing etoposide mediated apoptosis and caspase activity. Ectopic expression of human IAP genes inhibits apoptosis induced by a variety of different stimuli (Sanna et al., 2002).

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Figure 5. Mechanism of IAP action in the prevention of apoptosis. The apoptosis pathways activated by Fas, mitochondria, or TNFR-1 have been extensively characterized. IAP can block caspases-3, -7, and -9 by direct inhibition. Another possible mechanism for IAP action involves the interaction of these proteins with NF-kB and the JNK signal transduction pathway, which is activated by TRAFs through TNFR-1 and TNFR-2 type cytokine receptors. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Figure 6. The activation of caspase-9. Caspase-9 is activated in response to stimuli that triggers the release of cytochrome-c from the mitochondria. The coordinated action of caspase-9, dATP, APAF-1, and cytochrome-c leads then to the activation of other substrate proteins that are part of the caspase cascade.

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FLICE-inhibitory protein (FLIP)

FLIP, also named CLARP, FLAME-1, MRIT, and Casper, is an inhibitor of apoptosis and is predominantly expressed in muscle and lymphoid tissues. FLIP proteins are expressed as alternatively spliced variants of a single FLIP gene. All those variants contain a long pro-domain that harbors tandem DEDs. Following the pro-domain, FLIPs (with the exception of the short form FLIP) possess a caspase protease region, which is enzymatic inactive (Holcik and Korneluk, 2001).

The short form of FLIP contains two DEDs and is structurally related to the viral FLIP inhibitors of apoptosis, whereas the long-form, FLIP(L), contains an additional caspase-like domain in which the active-centre cysteine residue is substituted by a tyrosine residue. FLIPs and FLIP(L) interact with the adaptor protein FADD and the protease caspase-8, potently inhibiting apoptosis induced by all known human death receptors (Tschopp et al., 1998; Juo et al., 1999; Holcik and Korneluk, 2001).

Maedler et al. (2002a) observed decreased expression of FLIP in human pancreatic beta-cells of Type II diabetic patients. In vitro exposure of islets from non-diabetic organ donors to high glucose levels decreased FLIP expression and increased the percentage of apoptotic TUNEL-positive beta-cells; FLIP was no longer detectable in such TUNEL-positive beta-cells. Upregulation of FLIP, by incubation with the transforming growth factor beta or by transfection with an expression vector coding for FLIP, protected beta-cells from glucose-induced apoptosis, restored beta-cells proliferation, and improved beta-cells function. The beneficial effects of FLIP overexpression were blocked by an antagonistic anti-Fas antibody, indicating their dependence on Fas receptor activation. Overexpression of cFLIP protected mouse beta-cells against TNF-alpha-induced caspase-8 activation and apoptosis. This phenomenon has been shown to be associated with enhanced NF-kappaB transcriptional activity, suggesting that cFLIP may have an impact on the outcome of death receptor-triggered responses by directing the intracellular signals from beta-cell death to beta-cell survival (Cottet et al., 2002).

Dominant negative caspase

Several caspases are expressed as multiple isoforms by alternatively splicing the primary transcript. Caspases-1, -2, -3, -6, -7, and -8 are all detected in protein variants. Among the various isoforms in which they may be present, some have been shown to be enzymatically inactive variants that are expressed as modified mRNAs or truncated proteins. Those are believed to play a crucial role in the negative or positive regulation of caspase activity. Caspase-8, for example, is expressed in eight isoforms that differ by deletions or sequence variations in the N-terminal pro-domain (containing the DEDs) or by loss of the C-terminal part that normally encodes the p10/p20 caspase subunits. One of its isoforms (MACH alpha-3 isoform) has been clearly shown to have a dominant negative effect on the activity of the active caspase-8 enzyme and to provide effective protection against Fas-mediated apoptosis (Boldin et al., 1996).

Caspases-1, -8, -9, and -3 are situated at pivotal junctions in various apoptotic pathways
Caspase-1

Caspase-1 was the first member of the caspase family to be identified and originally labeled ICE (for IL1β-converting enzyme) as a novel type of cysteine protease responsible for the conversion of precursor IL1β to its mature form in monocytes. The mature form of IL-1beta, cleaved at Asp-116-Ala-117, is a key mediator of inflammation. It was discovered, based on the sequence similarity to the C. elegans death gene, ced-3. This initiated studies about its possible role in PCD. However, ICE knockout mice develop normally with no apparent physiological or morphological aberrations. This indicates that ICE has no substantial role in apoptosis (Fantuzzi and Dinarello, 1996).

The mature form of IL1β-converting enzyme is derived from a precursor of 404 amino acids, and it is generated by the removal of the N-terminal 119 amino acids sequence of an intermediate fragment spanning residues 298–316. The active form thus comprises a p20 (residues 120–297) and a p10 (residues 317–404) subunit, both of which are essential for activity (Alheim and Bartfai, 1998).

Human islets of Langerhans undergo apoptosis in response to IL-1beta and Fas ligation (Loweth et al., 1998). In vitro studies have demonstrated that IL-1beta decreases insulin and DNA contents in pancreatic islet beta-cells and causes structural damage by inhibiting glucose oxidation. This structural damage of the DNA induces apoptosis (Larsen et al., 1998). IL-1beta induced kinase activity toward Elk-1, activation transcription factor 2, c-Jun, and heat-shock protein 25 in rat islets. IL1β-induced rat pancreatic islet NO synthesis requires both the p38 and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases. Viral infection may initiate beta-cell damage during the development of autoimmune diabetes. The viral replicative intermediate dsRNA stimulates beta-cell production of pro-IL-1beta, and following cleavage to its mature form by IFN-gamma-activated ICE, IL-1 then initiates beta-cell damage in a NO-dependent fashion (Heitmeier et al., 2001).

Islets from NMRI mice cultured in the presence of IL-1beta show an induction in the expression of ICE. This result supports a direct role of the Fas/Fas-L and the perforin systems in the autoimmune destruction of insulin producing cells (Yamada et al., 1996; Ingelsson et al., 1998).

Caspase-8

Caspase-8 consists of 479 amino acids with a molecular weight of 55 kDa. There are eight alternative products derived from the alternative splicing of this gene. Like most of the upstream proteases, Caspase-8 mediates the activation cascade of caspases in the process of Fas mediated and TNF receptor-1 induced cell death. Caspase-8 contains two DED, which interact with the DED of FADD and result in the formation of a complex among Fas, FADD, and caspase-8. The resulting aggregate called the DISC performs caspase-8 proteolytic activation (Xerri et al., 1999; Kruidering and Evan, 2000). The active dimeric enzyme is then liberated from the DISC and free to activate downstream apoptotic proteases (Zhou et al., 1997; Slee et al., 1999a).

Two pathways for interaction of Fas and caspase-8 have been proposed (Juo et al., 1998). The first pathway involves the accessory protein FADD and the pro-enzymatic form of caspase-8. Upon oligomerization of the receptor molecules, the FADD molecule binds to Fas by homotypic interactions between its death domains. In the second pathway, the pro-enzymatic form of caspase-8 binds to the Fas/FADD complex by interaction between the DEDs of the FADD and the caspase-8 molecules. This results in the activation of caspase-8, which then triggers the caspase cascade. This pathway can be blocked by FLIP but not by Bcl-2 (Hennino et al., 2000).

Active caspase-8 can induce apoptosis by activating numerous caspases, inculding caspases-3, -4, -6, -7, -9, -10, and -13. PARP and Bid are substrates of caspase-8 as well.

Cytokine-induced apoptosis of betaTc-Tet (highly differentiated insulin-secreting cell line) has been also associated with the activation of caspase-8 (Cottet et al., 2002). In rat pancreatic acinar cells, cholecystokinin (CCK) has been shown to be capable of inducing caspase activation, cytochrome-c release, and mitochondrial depolarization. The mitochondrial dysfunction mediated by caspase-8 leads then to the activation of caspase-3 (Gukovskaya et al., 2002). Supramaximal secretory stimulation of acini with CCK leads to a rapid redistribution and activation of caspase-8, followed by degradation of the intermediate filament (IF) termed plectin that, in turn, promotes the breakdown of the cytoskeleton. Inhibition of caspase-8 before CCK hyperstimulation prevents plectin cleavage, stabilizes F-actin morphology, and reverses the inhibition of secretion (Beil et al., 2002).

Caspase-9

Caspase-9 (also termed APAF3, MCH6, APAF-3, and ICE-LAP6) is a member of the CED-3 subfamily and is constituted of 416 amino acids with a molecular weight 46 kDa. Caspase-9 has multiple mRNA forms produced by alternative splicing and is ubiquitously expressed. The highest level of caspase-9 mRNA has been observed in the heart. It is moderately expressed in the liver, skeletal muscle, and pancreas; and is present in very low levels in all other tissues.

Caspase-9 and Apaf1 bind to each other via their respective NH2- terminal ced-3 homologous domains in the presence of cytochrome-c and ATP (Henshall et al., 2001; Shiozaki et al., 2002) (Fig. 6). Compared to caspase-3, caspase-9 possesses a longer N-terminal pro-domain with high similarity to the pro-domains of CED-3 and caspase-2, which contain CARDs. The active pentapeptide domain of Caspase-9 differs from that of the other family members being QACGG instead of QACRG. The active caspases are tetramers formed by two large (usually ∼20 kDa) and two small (∼10 kDa) subunits. Residues from both types of subunits contribute to the formation of the substrate binding sites (two per tetramer). Small subunits are derived from the C-terminal portion of the zymogen polypeptide, by cleavage at one site, or two closely spaced sites on the C-terminal end of the active-site cysteine. Caspase-9 is an exception when compared to other caspases in that, in vivo, the active, mature enzyme retains the N-terminal pro-domain as part of the large subunit (35 or 37 kDa).

Activity of caspase-9 was detected by a fluorometric assay in Simvastatin-induced isolated human pancreatic islets (Contreras et al., 2002b). With treatment (up to 48 h) of HIT-T15 with 3 μg/ml MPA, a marked increase of caspase-2 activity (+343%) and moderate activation of caspase-9 (+150%) and caspase-3 (+145%) have been found (Huo et al., 2002).

Caspase-3, pro-caspase-9, caspase-7, and PARP are all the substrates of caspase-9. In the presence of dATP, caspase-9 is directly activated by Apaf-1 and cytochrome-c (Susin et al., 1997). Active caspase-9, in turn, activates caspase-3 and, by doing so, initiates the apoptotic machinery that leads to apoptosis (Fujita et al., 2001). Caspase-9 activity is regulated by protein phosphorylation (Cardone et al., 1998). The kinase Akt phosphorylates pro-caspase-9 at Ser196 and, by this, inhibits proteolytic processing of pro-caspase-9.

Homozygous caspase-9 knockout (Casp-9−/−) is lethal in most cases. Additionally, Casp-9−/− newborn mice have been shown to be consistently smaller than control littermates and most viable knockout mice die before postnatal day 3. Casp-9−/− mice displayed protrusions of the brain tissue from the skull. This has been shown to be associated with gross perturbations of brain structure that were most severe within the cortex and the forebrain (Hakem et al., 1998; Harrison et al., 2001). Interestingly, these gross morphological abnormailities have been shown to be remarkably similar to those observed in mice lacking caspase-3 (Zheng et al., 2000).

Embryonic stem (ES) cells from Casp-9−/− knockout mice have been shown to be resistant to a variety of apoptotic stimuli (e.g., UV, gamma irradiation, etoposide, cisplatinum, adriamycin). Thymocytes and lymphocytes develop normally in Casp-9−/− mice, but they are resistant to apoptosis induced by dexamethasone or gamma irradiation. Furthermore, Casp-9−/− thymocytes showed resistance to etoposide, but were sensitive to stimuli like anti-Fas, TNF-alpha, UV irradiation, or heat shock (Hakem et al., 1998; Kuida et al., 1998). These data support the notion that multiple apoptotic pathways are present in eukaryotic cells.

Caspase-3

Caspase-3 (also termed CPP32, apopain, and Yama) is a member of the IL-1β converting enzyme family of cysteine proteases and has been shown to have the highest sequence similarity to ced-3. The enzyme is composed of 17 and 12kDa subunits derived from a common proenzyme termed pro-caspase-3. Caspase-3 is widely distributed in eukarioric cells, with high expression in cell lines of lymphocytic origin (Fernandes-Alnemri et al., 1994).

Caspase-3 plays a major role in Fas-mediated apoptosis of beta-cells. (Yamada et al. (1999), using a beta-cell line transfected with human Fas, showed that the cross-linking of Fas by anti-Fas resulted in the elevation of caspase-3-like, but not caspase-1-like, protease activity 2–12 h after the addition of the anti-Fas. A caspase-3 inhibitor, Z-Asp-Glu-Val-Asp-fluoromethyl ketone, attenuated the Fas-mediated beta-cell apoptosis while a caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-chloromethylketone, failed to suppress the apoptosis. Thus, the Fas-induced death signal apparently bypassed caspase-1 in the cells. Furthermore, an anti-sense caspase-3 construct blocked caspase-3 activation and substantially suppressed Fas-triggered apoptosis of hFas/betaTC1 cells. These observations suggest the essential role of caspase-3 in Fas-mediated apoptosis of the beta-cell line.

Caspase-3 is activated during apoptotic signaling events by upstream proteases including caspases-6, -8, and cytotoxic T-cell derived granzyme-B (Zapata et al., 1998). The crystal structure of caspase-3 shows that the active enzyme is a tetramer, containing two small and two large subunits. A small and a large subunit form a heterodimer and two of these heterodimers form the mature tetramer (or homodimer of the two heterodimers).

Caspase-3 deficient mice die at 1–3 weeks of age with characteristic morphological brain-related aberrations (Keramaris et al., 2000). Neuronal apoptosis is markedly failed in these mice resulting in hyperplasias, ectopic cell masses, and duplicated brain structures. Interestingly, other major organs are uneffected by the mutation. These results indicate that caspase-3 has a non-redundant role in neural apoptosis, whereas in other tissues other isoforms can substitute for its loss.

Caspase-3 has been identified as being a key mediator of apoptosis of mammalian cells. It can cleave numerous substrates with a DXXD sequence to effect the morphological changes associated with apoptosis (Fattman et al., 2001). Caspase-3 substrates include: PARP, SREBs, gelsolin, caspases-6, -7, -9, DNA-PK, MDM2, Gas2, fodrin, b-catenin, lamins, NuMA, HnRNP proteins, topoisomerase I, FAK, calpastatin, p21Waf1, resenelin2, and DFF45 (human 45 kDa DNA fragmentation factor (DFF)), etc. The significance of PARP cleavage is not clear, although this phenomenon represents an excellent marker for caspase activation and the presumption of ongoing apoptosis (Kothakota et al., 1997).

FROM THE ACTIVATION OF CASPASES TO THE APOPTOTIC CHANGES IN THE MORPHOLOGY OF PANCREATIC BETA-CELLS

  1. Top of page
  2. Abstract
  3. APOPTOSIS IN PANCREATIC BETA-CELLS
  4. STIMULI OF APOPTOSIS IN PANCREATIC BETA-CELLS
  5. CASPASES: ACTIVATION AND PATHWAY
  6. FROM THE ACTIVATION OF CASPASES TO THE APOPTOTIC CHANGES IN THE MORPHOLOGY OF PANCREATIC BETA-CELLS
  7. STRATEGIES FOR PREVENTING APOPTOSIS OF ISLETS
  8. CONCLUSION
  9. Acknowledgements
  10. LITERATURE CITED

The activation of the caspase cascade is mediated by structural and conformational changes of multiple intracellular substrates (Wang and Wang, 1999; Saraste and Pulkki, 2000; Wride, 2000). These include: (a) changes of the gene expression profile and of metabolic pathways utilized; (b) activation of specific signaling pathways; (c) collapse of the cytoplasm and nuclear membranes, degradation of mitochondrial and nuclear DNA; (d) maintenance of the intact cell membranes; (e) preventing release of inflammatory factor; (f) engulfing of the dying cell by adjacent cells. All the functional and morphological changes listed here above occurs in a sequential fashion, such that three distinct stages leading to cell apoptosis could be identified.

First stage: genetic reprogramming

This is a stress response stage characterized by genetic reprogramming. In this stage, the protein profile changes; there is the expression of stress proteins, caspases and cellular concentration of Ca2+ are increased, while polyamines are decreased (Ha et al., 1997; Duchen, 2000).

The NO-induced beta-cell death is the typical apoptosis. Three different NO donors (SNAP, NOR3, and NOC7) induced apoptosis in a beta-cell line, MIN6 cells, in a concentration-dependent manner. SNAP, at 200 μM, increased cytosolic Ca2+ concentration ([Ca2+]i) and further induced apoptosis. This effect was blocked by a Ca2+ chelator, BAPTA-AM, and by an inhibitor of a Ca2+-dependent protease, calpain. These demonstrated that excessive NO production induces apoptosis, wherein an increase in [Ca2+]i and resultant activation of calpain play a key role (Sjoholm et al., 2001). Whereas, alterations in cytosolic Ca2+ homoeostasis do have a significant role in certain forms of beta-cell death, Bai et al. demonstrated they do not contribute to the pathway of apoptosis evoked by hA in islet beta-cells (Nakata et al., 1999).

Polyamines are also involved in the apoptotic process. Cultured islets from lean and obese animals contained significantly less polyamines than freshly isolated islets. Spermine-to-spermidine ratio was elevated in freshly isolated islets from young obese mice compared with those from lean mice. In islets from old obese animals, spermidine content was decreased, whereas the content of spermine was not different from that of young obese mice (Bai et al., 1999).

Sometimes, light chromatin changes could be also seen in the nucleus (Mikhailov et al., 2001). Apoptosis signal pathways have been started in this stage, but this process is reversable with the withdrawal of apoptotic stimuli.

Second stage: formation of blebbing and FLP

This stage is characterized by the activation of many endonucleases and by changes in the cell metabolism. Two peculiar morphological changes may also take place. They are termed blebbing and phosphatidylserine flip or FLP. Blebbing occurs when active initiator caspases cleave cytoskeletal and other related proteins (including vimentin, actin, and fodrin), leading to the formation of blebbing on the cell surface (Martin et al., 1995; Mills et al., 1998; Deschesnes et al., 2001; Grzanka, 2001) and thus predisposing to the shrinkage of the cell.

Aminophospholipids, such as phosphatidylserine or phosphatidylethanolamine, are normally confined to the inner leaflet of the plasma membrane while the outer leaflet contains mainly neutral phospholipids such as phosphatidylcholine (Middelkoop et al., 1989; Rice, 1998). This asymmetry in membrane aminophospholipid composition is achieved by the activity of enzymes such as the ATP-dependent aminophospholipid translocase (flippase) that preferentially transports phosphatidylserine and phosphatidylethanol-amine from the outer to the inner leaflet (Fabisiak et al., 1998). Active translocase counteracts spontaneous flipping of phosphatidylserine to the outer leaflet by relocating it to the inner leaflet. In the apoptosis process, the activation of the initiator caspase complex results in cleavage of translocase (flippase) and/or activation of scramblase (floppase). This is followed by a subsequent flip of phosphatidylserine from the inner to the outer leaflet of the plasma membrane (Emoto et al., 1997; Verhoven et al., 1999).

The 37-amino acid polypeptide amylin is the principal constituent of the amyloid deposits that form in the islets of Langerhans in patients with Type II diabetes mellitus, but its role in the pathogenesis of this disease is unresolved. Saafi et al. (2001) investigated the time-dependent morphological and ultrastructural changes in 10 microm HA-treated cultured RINm5F islet beta-cells. Membrane blebbing and microvilli loss were the earliest detectable apoptosis-related phenomena, already evident 1 h after HA exposure.

Annexin-V is a Ca2+-dependent phospholipid-binding protein with high affinity for phosphotidylserine (PS), hence, this protein can be used as a sensitive probe for PS exposure upon the outer leaflet of the cell membrane and be used for the detection of apoptotic cells. The simultaneous application of propidium iodide as a DNA stain, used for dye exclusion tests, allows annexin-V positively stained cell clusters to be distinguished from necrotic cells. FACS analysis has been used in determining the apoptosis of Min6 induced by H2O2 (Hui et al., 2003). Measurement of caspase-3 activity and annexin V binding analysis might represent reliable markers of the early events of islet cell apoptosis (Lorenzo et al., 1994).

In this stage, in the presence of stimuli, apoptotic signals are passing through the specific pathway and changing the balance point between pro-apoptosis and anti-apoptosis as discussed above. Apoptosis early in this stage is still considered “reversible,” but it is “irreversible” in the later stage of the phase.

Third stage: protein crosslinking and apoptotic body formation

This is the last stage of apoptosis and is characterized by a protein crosslinking process, which is led by TG, and is associated with the formation of apoptotic bodies and the phagocytosis of apoptotic bodies (Tucholski and Johnson, 2002). The typical nuclear changes include chromatin condensation and alterations of nuclear shape (Dobrucki and Darzynkiewicz, 2001). These changes are regulated by a broad array of proteins critical for cell survival and activated by execution caspases (Susin et al., 2000; Slee et al., 2001) (Table 3). Some of the typical substrates affected by execution caspases include: IFs (including keratins-15, -17, -18, and -19); poly-(ADP) ribose polymerase (PARP); topoisomearse-I; DNA-dependent protein kinase (DNA-PK); IACD; T-cell restricted intracellular antigen-related protein (TIAR); and TG-II. At this stage, apoptosis cascades have been fully activated and the effects of caspases on substrates result in “irreversible” cell damage.

Table 3. Substrates for caspases action
ClassificationSubstrates
Cell death proteinBcl-2; Bcl-xL; Bid; CrmA; IAP; p28 Bap31; p35; pro-caspases
Cell cycle regulationcdc27; cyclin A; MDM2; p21(Cip1/Waf1); p27(kip1); PISLRE kinase; Rwtinoblatama protein; Wee1 phosphatase
CytoskelatonActin; beta-catenin; Gas2; gelsolon; keratin-18 and -19; lamins; plakoglobin
Transcription factorsHeat-shock factor; GAT-1; IkB-alpha; NF-kB(p50, p65) NRF-2; Sp1; STAT1; sterol-regulatory element-binging protein
DNA metabolismAcinus; DNA-dependent protein kinase (DNA-PK); DNA replication complex C (DSEB/RFC140); ICAD; MCM3DNA replication factor; NuMA; PARP; topoismerase 1
RNA metabolismEukaryotic initiation factor 2 alpha; heteronucear ribonuclear proteins C1 and C2; 70 kDa U1-snRNP
Cytokine precursorsPro-IL-1beta; Pro-IL-16; Pro-IL-18 (IGIF)
Signal transductionAdenomatous polyposis coli protein (APC); Akt/Pkb; calmodulin-dependent kinase IV; c-Raf; D4-GDP dissociation inhibitor; Eyn tyrosine kinase; focal adhesion kinase; MEKK1 MST/Ksr; PAK-2/hPAK65; protein kinase c delta; protein kinase C theta; protein kinase C-related kinase 2; protein phosphatase 2A; ras GTPase activating protein; TCR-z chain
OthersCalpastatin; Hsp90; Nedd4; phosphalipase A2; rabaptin-5; transglutaminase
IFs

IF proteins represent one of the most important type of substrates for caspase activity (Prasad et al., 1999). They are thin filaments (actin) and microtubules that frequently work together to enhance both structural integrity, cell shape, and cell and organelle motility. IFs are stable and durable. IFs are prominent in cells that withstand mechanical stress and are the most insoluble part of the cell. There are five different types of IFs. Types I and II are acidic keratin and basic keratin, respectively, and are produced by different types of epithelial cells (bladder, skin, etc.). Type III includes a variety of proteins distributed in a number of cell types; among those, vimentin is expressed in fibroblasts, endothelial cells, and leukocytes; desmin in muscle; glial fibrillary acidic factor in astrocytes and other types of glia; and peripherin in peripheral nerve fibers. Type IV inculdes neurofilament-H (heavy), -M (medium), and -L (low), as defined on the basis of their molecular weight. Another member of the Type IV group is “internexin” and some non-standard IV's are found in lens fibers of the eye (filensin and phakinin). Type V proteins are the lamins, which have a nuclear signal sequence so they can form a filamentous support inside the inner nuclear membrane. Lamins are vital to the re-formation of the nuclear envelope after cell division (Omary et al., 1998). Apoptosis-induced cleavage of IF proteins has been previously demonstrated for keratins-15, -17, -18, -19 (Prasad et al., 1998; Ku and Omary, 2000), for lamin-A, -B1, -B2, -C, and vimentin (Steen and Collas, 2001).

Recently, a protein of 65–70 kDa protein isolated from intact rat islets and clonal beta (HIT or INS) cells has been immunoprecipitated using lamin-B anti-serum. Incubation of purified HIT cell-nuclear fraction with [(3)H]S-adenosyl methionine yielded a single carboxyl methylated protein peak (approximately 65–70 kDa); this protein was immunologically identified as lamin-B. Several methylation inhibitors, including acetyl farnesyl cysteine (a competitive inhibitor of protein prenyl cysteine methylation), inhibited the carboxyl methylation of lamin-B. This indicates that the carboxyl-methylated amino acid is cysteine. These findings, together with other recent observations demonstrating that inhibition of protein isoprenylation causes apoptotic death of the pancreatic beta-cells, raise an interesting possibility that inhibition of C-terminal cysteine modifications of lamin-B might result in disruption of nuclear assembly. This disruption would then lead to further propagation of apoptotic signals, including DNA fragmentation and chromatin condensation (Kowluru, 2000).

Poly-(ADP-ribose) polymerase (PARP)

PARP is a nuclear enzyme activated by binding to a single- or double-strand break of DNA. This enzyme modifies various nuclear proteins by poly-(adp-ribosyl)ation and is involved in the regulation of various important cellular processes such as differentiation, proliferation, and tumor transformation. It also regulates the molecular events involved in the recovery of cell after DNA damage (Tong et al., 2001; Uchida et al., 2001).

The molecular weight of PARP is 116 kDa. It is cleaved during apoptosis by caspase-3 into a 24-kDa fragment containing the DNA binding domain and an 89-kDa fragment containing the catalytic and automodification domains. The 24-kDa fragment irreversibly binds to DNA, contributing to the irreversibility of apoptosis by blocking the access of DNA repair enzymes to damged DNA (Boulares et al., 1999; Nargi-Aizenman et al., 2002).

Elevated concentrations of glucose have been shown to be associated with the production of reactive nitrogen and oxygen species, in vitro and in vivo. These agents may then trigger DNA single-strand breakage, which induces rapid activation of PARP. PARP, in turn, depletes the intracellular concentration of its substrate, NAD+, slowing the rate of glycolysis, electron transport, and ATP formation. This process is believed to play a fundamental role in the acute endothelial dysfunction observed in diabetic blood vessels (Soriano et al., 2001).

The nicotinamide-induced apoptosis in insulin producing cells is associated with cleavage of poly(ADP-ribose) polymerase. Nicotinamide-exposed cells show a high mitotic rate and an inhibition of ADP-ribosylation. These events lead to the release of PARP bound to DNA strand breaks and results in the activation of PCD (Saldeen and Welsh, 1998; Kolb and Burkart, 1999).

Topoisomerases

DNA topoisomerases are a class of enzymes involved in the regulation of DNA supercoiling. Type I topoisomerases change the degree of supercoiling of DNA by causing single-strand breaks and re-ligation, whereas Type II topoisomerases cause double-strand breaks (Bailly, 2000). Various observations indicate that topoisomerase I is a substrate for both caspase-3 and caspase-6. The interaction with caspase-3 leads to the formation of an 80-kDa major fragment and a 76-kDa minor fragment, while the interaction with caspase-6 predominantly generates a 76-kDa fragment and a 82-kDa fragment (Samejima et al., 1999).

DFF

Chromosome DNA fragmentation into nucleosomal multimers is a main hallmark of apoptosis and is mediated by DFF. DFF is a heterodimeric protein complex consisting of DFF40 and DFF45. The DFF45 subunit is located in the cell cytoplasm. A mouse homologue of human DFF has been identified as a DNase inhibitor designated ICAD. Upon cleavage of DFF/ICAD, a caspase activated deoxyribonuclease (CAD) is released and activated and eventually causes the degradation of DNA in the nuclei (Enari et al., 1998; Sakahira et al., 1998; Wyllie, 1998).

DFF has been found in a variety of apoptotic events. Following 6–12 h of HA-treatment in RINm5F pancreatic islet beta-cells, chromatin margination became evident, consistent with detecting DNA laddering about the same time. Nuclear shrinkage, nuclear membrane convolution, and prominent cytoplasmic vacuolization were clearly recognized at 22 h post-treatment (Saafi et al., 2001). DNA fragmentation could be detected early in islet cells after isolation and the beta-cells were primarily involved (Paraskevas et al., 2000). IL-1β and other cytokines have been demonstrated to be capable of inducing a decrease of insulin content and to induce DNA fragmentation of rat pancreatic islet beta-cells (Hadjivassiliou et al., 1998; Papaccio et al., 2002). The introduction of the potent immunosuppressive drugs tacrolimus (FK) and cyclosporine (CSA) has markedly improved the outcome of solid organ transplantation. However, these drugs can cause post-transplantation diabetes mellitus. Electron microscopy showed cytoplasmic swelling and vacuolization, and a marked decrease or absence of dense-core secretory granules in beta-cells; the changes were more pronounced in patients on FK (Drachenberg et al., 1999).

Caspase-3 cleaves the 45-kD subunit at two sites to generate an active factor (Liu et al., 1997). When DFF-45 is cleaved and activated, caspase-3 activity is no longer required for DNA fragmentation, although it is necessay for the cleavage of other nuclear substrates such as laminin B1 and poly-(ADP-ribose) polymerase (PARP).

TIAR

T-cell-restricted intracellular antigen-related protein; or TIAR is an RNA-binding protein of 42 kDa. TIAR mRNA transcripts are observed in a wide variety of cell types and encode two isoforms (42 and 50 kDa) by different splicing of the same gene. TIAR is concentrated in the nucleus of hematopoietic and non-hematopoietic cells. TIAR can trigger DNA fragmentation in permeabilized thymocytes (Kawakami et al., 1992). In Fas-induced apoptosis, TIAR moves from the nucleus to the cytoplasm. Redistribution of TIAR precedes the onset of DNA fragmentation and is a specific consequence of nuclear disintegration. Cytoplasmic redistribution of TIAR is not observed during cellular activation triggered by mitogens such as concanavalin A or phytohemagglutinin (Taupin et al., 1995). Increased expression of TIAR has been observed in patients with Alzheimer's disease, suggesting that TIAR may be related to the process of neurodegeneration in the hippocampus (Oleana et al., 1998).

No report about the investigation of TIAR in islets has been found yet.

TG-II

Tissue TG or Type II TG (tTG) is a Ca2+-dependent enzyme catalyzing an acyl transfer reaction in which new g-amide bonds are formed between a g-carboxamide group of peptide-bound glutamine residues and various primary amines. Tissue TG is a 75-kDa cytosolic monomeric globular protein expressed in the majority of cells and tissues and is translated as a fully active enzyme. This enzyme has a wide variety of functions including modeling of the extracellular matrix, stimulus-secretion coupling, receptor-mediated endocytosis, cell differentiation, and tumor growth (Aschoff et al., 2000; Dvorcakova et al., 2002).

Transglutaminases catalyze the posttranslation modification of proteins by catalyzing a Ca2+-dependent acyl-transfer reaction resulting in the formation of new g-amide bonds between g-carboxamide groups of peptide-bound glutamine residues and various primary amines. Such glutamine residues serve as an acyl-donor and the most common acyl-acceptors are e-amino groups of peptide-bound lysine residues or primary amino groups of some naturally occurring polyamines, like putrescine or spermidine. The active site of cysteine reacts first with the g-carboxamide group of glutamine residue to form the acyl-enzyme intermediate under the release of ammonia. In the second step, the complex reacts with a primary amine to form an isopeptide bond and liberate the reactivated enzyme. TG was found to be involved in the islet membrane-mediated events necessary for glucose-stimulated insulin release (Bungay et al., 1986). Its activity in rat islet homogenates was increased after preincubation of the islets at high glucose concentration (Gomis et al., 1986). TG-catalyzed cross-linking of proteins phosphorylated has been confirmed in the intact glucose-stimulated pancreatic beta-cell (Owen et al., 1988).

Although tTG was originally thought to be responsible for the protein crosslinking (which prevents the leakage of intracellular components, thereby reducing inflammation and autoimmunity), recent evidence indicates that tTG is a multifunctional enzyme. Indeed, tTG has been shown to be involved in the complex upstream regulation of the apoptotic machinery (Melino and Piacentini, 1998; Aschoff et al., 2000), acting as a GTP-binding protein capable of signaling properties and capable of binding/crosslinking specific cytosolic and nuclear substrates. The cytosolic level of Ca2+, GTP, S-nitrosylation, and polyamines regulates the function of tTG. Immunohistological studies, together with the isolation of highly polymerized protein products from apoptotic cells, have established tissue TG as a useful biochemical marker of PCD.

STRATEGIES FOR PREVENTING APOPTOSIS OF ISLETS

  1. Top of page
  2. Abstract
  3. APOPTOSIS IN PANCREATIC BETA-CELLS
  4. STIMULI OF APOPTOSIS IN PANCREATIC BETA-CELLS
  5. CASPASES: ACTIVATION AND PATHWAY
  6. FROM THE ACTIVATION OF CASPASES TO THE APOPTOTIC CHANGES IN THE MORPHOLOGY OF PANCREATIC BETA-CELLS
  7. STRATEGIES FOR PREVENTING APOPTOSIS OF ISLETS
  8. CONCLUSION
  9. Acknowledgements
  10. LITERATURE CITED

As we discussed above, apoptosis can occur in both Type I and II diabetes, as well as in islet transplantation. So it not only plays an essential role in diabetic pathology, but is also critical for diabetes treatment. The advancement in understanding of the apoptotic cascade of pancreatic islets provides a solid base for developing new strategies to prevent their apoptosis. The general rules of the stategies include: (1) to remove stimuli from the islet and keep it in a safety environment; (2) to attenuate the apoptotic stimuli and stop the apoptosis at an early stage; (3) to block the pathway of apoptosis and stop the process; (4) to change the balance between pro- and anti-apoptosis and reverse the cellular apoptotic process. To date, many methods have been successfully reported to be capable of preventing beta-cell apoptosis, some of which appear promising for a potential clinical application.

Scavenging-free radicals

High glucose and cytokines induce the production of free radicals. These kinds of molecules can further damage beta-cells. For example, IL-1beta, the main apoptotic inducer in production of free radicals, can induce free oxygen, lipid hydroperoxide, NO, and peroxynitrite production. Scavengers of free radicals have been demonstrated to be helpful in preventing apoptosis of islets. Imidazoline compounds (RX871024 (RX)) protected beta-cells against damage caused by IL-1beta-induced free oxygen and nitrogen radicals (Papaccio et al., 2002). Pretreatment of beta-cells (RINm5F) with gamma-tocopherol (but not alpha-tocopherol) afforded partial protection against the inhibitory effects of NO, whereas specifically inhibiting inducible NO synthase with N-nitro-l-arginine completely reversed the IL-1beta effects (Sjoholm et al., 2000). Expression of calbindin-D, a 28-kDa calcium binding protein, decreases the production of lipid hydroperoxide, NO, and peroxynitrite production in pancreatic islet beta-cells and protects them from cytokine-induced apoptosis and necrosis (Rabinovitch et al., 2001).

Metalloporphyrin-based SOD mimics scavenge ROS and protects cells from oxidative stress and apoptosis. The SOD mimic significantly inhibits antigen-presenting cell-dependent T-cell proliferation and IFN-gamma production in vitro. In addition, pretreatment of lipopolysaccharide (LPS)-stimulated peritoneal macrophages with SOD mimic inhibited the LPS-dependent increase in TNF-alpha as well as the NADPH oxidase-dependent release of superoxide. This compound also protected NIT-1 insulinoma cells from IL1β and alloxan cytotoxicity in vitro (Piganelli et al., 2002).

Heme oxygenase-1 (HO-1) has been described as an inducible protein capable of cytoprotection via radical scavenging and apoptosis prevention. HO-1 upregulation was induced reproducibly with protoporphyrins and was correlated with protection from apoptosis induced in vitro with proinflammatory cytokines or Fas engagement. Furthermore, in vivo, HO-1 upregulation resulted in improved islet function in a model of marginal mass islet transplantation in rodents (Pileggi et al., 2001; Tobiasch et al., 2001).

Blocking the Fas and FasL pathway

Fas/Fas ligand interactions play an essential role in the initiation of murine autoimmune diabetes (Nakayama et al., 2002). Beta-cells from FasL transgenic NOD mice are more susceptible to cytokine-induced apoptosis than wild-type beta-cells. This is supported by the hypothesis that if beta-cells express FasL, then Fas-FasL interactions on the beta-cells surface are able to mediate beta-cells self-death in the absence of T-cells. Interventions that block the Fas-FasL pathway may be useful, therefore, in the prevention or treatment of Type 1 diabetes (Petrovsky et al., 2002). Indeed, Fas antibody, FasL antibody, or any other reagents that could decrease the expression and translocation of Fas could be used in blocking apoptosis of an islet. Nakayama et al. (2002) demonstrated that administration of anti-FasL antibody at 2–4 weeks of age completely prevented insulitis and diabetes. High glucose-induced apoptosis is due to the interaction between the constitutively expressed FasL and the upregulated Fas. Maedler et al. (2001) found that the effect of high glucose was blocked by an antagonistic anti-Fas antibody). Klein et al. (2000) developed anti-Fas ribozyme and showed that by using this approach, they were able to inhibit Fas-mediated apoptosis in mouse insulinoma betaTC-3 cells. Augstein et al. (2002) found that inflammatory insults specifically induce translocation of Fas to the beta-cell surface and that interference with this cell surface Fas expression is a new strategy to improve beta-cell survival in inflamed islets.

Inducing peripheral T-cell apoptosis via the Fas-FasL pathway is another potential method for reversal of autoimmune diabetes. Administration of the Fas agonist immediately after onset of diabetes led to reversal of diabetes in NOD mice (Dharnidharka et al., 2002). Tian et al. (2001) found that LPS-activated B-cells, but not control B-cells, express Fas ligand and secrete TGF-beta. Co-incubation of diabetogenic T-cells with activated B-cells in vitro leads to the apoptosis of both T- and B-lymphocytes. Transfusion of activated B-cells, but not control B-cells, into prediabetic NOD mice inhibited spontaneous Th1 autoimmunity, but did not promote Th2 responses to beta-cells autoantigens. Co-transfer of activated B-cells with diabetogenic splenic T-cells prevented the adoptive transfer of Type I diabetes mellitus to NOD/scid mice. These findings provide new insights into T–B-cell interactions and may aid in the design of new therapies for human Type I diabetes mellitus.

Tumor cells can evade immune attack by killing lymphocytes through the expression of FasL on the tumor cell surface. The expression of FasL has been demonstrated in pancreatic cancer cell lines and in tissues. Co-culture of FasL and Fas-expressing PANC-1 cells and Fas-sensitive Jurkat cells showed that PANC-1 cells induced apoptosis of Jurkat cells, whereas the PANC-1 cells themselves and Jurkat cells cultured without the presence of PANC-1 showed few apoptotic changes. These findings suggest that FasL may play an important role in the ability of PDCs to escape from immune surveillance through the induction of apoptosis in tumor-attacking lymphocytes (Satoh et al., 1999).

Blocking the caspase-signaling pathway

Using freshly isolated islets, three major mechanisms have been determined to be involved in early events leading to cell apoptosis (II-APO): (i) caspase-6 activation; (ii) TRAIL-induced apoptosis, and (iii) mitochondrial-associated apoptosis. Thomas et al. (2001) showed that inhibition of these II-APO pathways improved isolated islet survival and reduced functional islet mass loss. It seems the importance of each caspase varies in diffenent models apoptosis. Z-Asp-Glu-Val-Asp-fluoromethyl ketone, an inhibitor of Caspase-3, inhibted the apoptosis of mouse beta-cells transfected with human Fas (Yamada et al., 1999). A specific caspase-2 inhibitor (Z-VDVAD-FMK), but not a caspase-3 inhibitor (DEVD-CHO), was also capable of restoring cell viability of HIT-15 cells treated with MPA (Huo et al., 2002). II-APO was exaggerated by the addition of the TRAIL mechanism. The TRAIL mechanism–induced II-APO was blocked by the caspase-6 inhibitor, VEID (Thomas et al., 2001).

PARP activity controls early steps of apoptosis and is another checking point to interfere with the caspase cascade. 5-iodo-6-amino-1,2-benzopyrone (INH(2)BP) (a potent inhibitor of PARS) has been found to be able to inhibit apoptosis of beta-cells induced by cytokines, peroxynitrite, and streptozotocin, both in vitro and in vivo (Mabley et al., 2001). Nicotinamide-c is a powerful PARP inhibitor. The suppression of PARP activity by nicotinamide not only decreases consumption of NAD+, the substrate of PARP, but also has major regulatory effects on gene expression, as shown for the major histocompatibility complex class II gene. The possible suppression of ADPRTs by nicotinamide would also affect CD38, a membrane-bound external ADP-ribosyl transferase with potent immunoregulatory properties (Kolb and Burkart, 1999).

Increasing the expression of pro-survival molecules and decreasing the pro-apoptosis molecules in beta-cells

Pro-survival members of the Bcl-2 class include Bcl-2 itself and Bcl-XL, while pro-apoptotic proteins include Bax, Bad, and Bid. Gene transfer of Bcl-2 has been shown to protect PI from apoptosis and necrosis in several models. The TRAIL mechanism–induced II-APO was blocked by ex vivo gene transfer of Bcl-2 (Thomas et al., 2001). Gene transfer with Bcl-2 confers cytoprotection to isolated adult porcine pancreatic islets when exposed to xenoreactive antibodies and complement (Contreras et al., 2001a,b). Overexpression of human Bcl-2 in beta TC1 cells partially protected them from cytokine-induced (IL-1beta, TNF-alpha, and IFN-gamma) cell death (Iwahashi et al., 1996). This transgenic overexpression of human Bcl-2 in islet beta-cells inhibits apoptosis but does not prevent autoimmune destruction (Allison et al., 2000).

The protective role of Bcl-x(L) has also been demonstrated. Although, overexpression of Bcl-x(L) in transgenic mice results in a reduced glucose-induced insulin secretion and hyperglycemia due to a defect in mitochondrial nutrient metabolism and in intracellular signaling for insulin secretion (Zhou et al., 2000).

Thomas et al. (2002) found that isolated human islets express Bax at a higher level compared with Bcl-2, suggesting the balance between pro-survival and pro-apoptosis molecules is one of the main mechanisms for islet cell death by apoptosis. It is possible that reducing islet expression of Bax, or regulating its activation, will help preserve islet cell mass after islet transplantation.

Increasing the expression of apoptotic inhibitors—FLIP

It has been shown that the caspase-8 inhibitor FLIP may divert Fas-mediated death signals into those for cell proliferation in lymphatic cells. Maedler et al. (2002a) observed expression of FLIP in human pancreatic beta-cells of non-diabetic individuals, which was decreased in tissue sections of Type 2 diabetic patients. Upregulation of FLIP protected beta-cells from glucose-induced apoptosis restored beta-cells proliferation, and improved beta-cells function. The beneficial effects of FLIP overexpression were blocked by an antagonistic anti-Fas antibody, indicating their dependence on Fas receptor activation.

Interfering with the signal pathway activating NF-kB

Nuclear factor kB (NF-kB) is a nuclear transcription factor that regulates expression of a large number of genes that are critical for the regulation of apoptosis, viral replication, tumorigenesis, inflammation, and various autoimmune diseases. The activation of NF-kB is thought to be part of a stress response, as it is activated by a variety of stimuli that include growth factors, cytokines, lymphokines, UV, pharmacological agents, and stress. In its inactive form, NF-kB is sequestered in the cytoplasm, bound by members of the IkB family of inhibitor proteins, which include IkBa, IkBb, IkBg, and IkBe. The various stimuli that activate NF-kB cause phosphorylation of IkB, which is followed by its ubiquitination and subsequent degradation. This results in the exposure of the nuclear localization signals (NLS) on NF-kB subunits and the subsequent translocation of the molecule to the nucleus. In the nucleus, NF-kB binds with a consensus sequence (5′-GGGACTTTCC-3′) of various genes and thus activates their transcription. IkB proteins are phosphorylated by an IkB kinase complex consisting of at least three proteins; IKK1/IKKa, IKK2/IKKb, and IKK3/IKKg. These enzymes phosphorylate IkB, leading to its ubiquitination and degradation. TNF binds to its receptor and recruits a protein called TNF receptor death domain (TRADD). TRADD binds to the TNF receptor-associated factor 2 (TRAF-2) that recruits NF-kB-inducible kinase (NIK). Both IKK1 and IKK2 have canonical sequences that can be phosphorylated by the MAP kinase NIK/ME KK1 and both kinases can independently phosphorylate IkBa or IkBb.

Islet graft injury by cytokines released from inflammatory cells (macrophages) that infiltrate the transplant site is mediated by NF-kappaB-dependent upregulation of iNOS gene expression and increased NO (NO) production by the islet. Baker et al. (2001) transfected the parent line with a dominant negative inhibitor of NF-kappaB. When treated with cytokine, 2Bm demonstrated significantly less NF-kappaB nuclear translocation, nitrite production, and apoptosis than parent MIN6. The rate of apoptosis in cytokine-treated 2Bm was a third less than that for cytokine-treated MIN6. Estradiol also has a protective role for isolated human islets. It acts against pro-inflammatory cytokine-induced cell death by lowering NF-kappaB nuclear translocation, cytochrome release, and caspase-9 activation (Contreras et al., 2002a). Similarly, Giannoukakis et al. (2000a) found that Fas-triggered apoptosis of human islets with IkappaB was inhibited following IkappaB alpha gene transfer.

TRAF-2 also interacts with A20, a zinc finger protein whose expression is induced by agents that activate NF-kB. A20 functions to block TRAF2-mediated NF-kB activation. A20 also inhibits TNF and IL-1 induced activation of NF-kB, suggesting that it may act as a general inhibitor of NF-kB activation. 1,25-(OH)2D3 was able to induce and maintain high levels of A20 and further protected RINm5F and human islet cells against cytokine-induced apoptosis (Riachy et al., 2002).

Simvastatin induces activation of the serine-threonine protein kinase AKT and increases survival of isolated human pancreatic islets (Contreras et al., 2002b). Glucose has been reported to promote pancreatic islet beta-cell survival through a PI 3-kinase/Akt-signaling pathway (Srinivasan et al., 2002).

The combination of IL-1beta and IFN-gamma increased both apoptosis and necrosis in rat islet cells. SB203580, but not the extracellular signal-regulated kinase inhibitor PD98059, partially prevented cytokine-induced apoptosis (Saldeen et al., 2001).

Improving beta-cell status with growth factors and/or other reagents

Many growth factors, characteristically known for their pro-proliferative properties and/or their action on cell differentiation, have also been shown to be capable of interfering with the sequence of events leading to cell apoptosis. The following growth factors have been investigated on their ability of inducing, delaying, or preventing PCD in vitro: TGF-alpha, bFGF, TGF-beta1, brain-derived neurotrophic factor (BDNF), aFGF, bFGF, insulin-like growth factor-I (IGF-1), PDGF, and HGF.

Many studies showed IGF-1 is a useful factor for protecting beta-cells from apoptosis. Pretreatment of neonatal rat islets of Langerhans with insulin (25 ng/ml) or insulin-like growth factor-110(-8)M) gave only partial protection against cell killing, but prevented the Fas-mediated component (Harrison et al., 1998). Beta-cells expression of IGF-I leads to recovery from Type I diabetes (George et al., 2002). IGF-I gene transfer prevented IL-1beta-induced, Fas-mediated apoptosis (Giannoukakis et al., 2000b). Using an arginine analog in culture or IGF-I pretreatment of islets, Mabley et al. (1997) demonstrated that the anti-apoptosis effect was carried out by its decreased cytokine induction of NO synthase in islets.

IGF-II has been shown to act as a beta-cell survival factor in vitro. Hill et al. (2000) examined whether IGF-II regulates beta-cell apoptosis in vivo. An IGF-II transgenic mouse model was used in which mouse IGF-II is overexpressed in skin, gut, and uterus driven by a keratin promoter so that circulating IGF-II is retained postnatally. The results show that a persistent presence of circulating IGF-II postnatally alters endocrine pancreatic ontogeny in the mouse and largely prevents the wave of developmental apoptosis that precipitates beta-cell turnover in neonatal life.

Islet beta-cell apoptosis was significantly decreased, whereas apoptosis of graft-infiltrating leukocytes was significantly increased in Ad TGF-beta1-transfected islet grafts. These observations demonstrate that overexpression of TGF-beta1, by gene transfection of NOD mouse islets, protected islet beta-cells from apoptosis and autoimmune destruction and delayed diabetes recurrence after islet transplantation (Grewal et al., 2002; Suarez-Pinzon et al., 2002).

Glucagon-like peptide-1 (GLP-1) is a hormone involved in regulation of insulin secretion. Farilla et al. (2002) demonstrated that GLP-1 promotes islet cell growth and inhibits apoptosis in Zucker diabetic rats. Furthermore, Hui et al. (2003) demonstrated that activation of the GLP-1 receptor inhibits H2O2-induced apoptosis in a cultured mouse insulinoma cell line (termed MIN6). This anti-apoptotic action of GLP-1 is mediated by a cAMP and PI3K-dependent signaling pathway.

Leptin increased the viability of isolated rat pancreatic islets by suppressing apoptosis (Okuya et al., 2001). Carbon monoxide protected isolated pancreatic islet cells from apoptotsis. This protection is mediated through guanylate cyclase activation, generation of cyclic GMP (cGMP), and activation of cGMP-dependent protein kinases (Gunther et al., 2002).

Gamma interferon (IFN-gamma) has been thought to play an important role in the pathogenesis of diabetes, but rIFN-gamma inhibits the diabetic process in NOD mice by decreasing anti-islet effector activity and, in turn, decreasing insulitis and islet destruction. The suppression of Th1 cell-related cytokines and/or augmentation of the macrophage cytokine IL-1 may play a role in the diabetes sparing effect of rIFN-gamma (Sobel et al., 2002).

CONCLUSION

  1. Top of page
  2. Abstract
  3. APOPTOSIS IN PANCREATIC BETA-CELLS
  4. STIMULI OF APOPTOSIS IN PANCREATIC BETA-CELLS
  5. CASPASES: ACTIVATION AND PATHWAY
  6. FROM THE ACTIVATION OF CASPASES TO THE APOPTOTIC CHANGES IN THE MORPHOLOGY OF PANCREATIC BETA-CELLS
  7. STRATEGIES FOR PREVENTING APOPTOSIS OF ISLETS
  8. CONCLUSION
  9. Acknowledgements
  10. LITERATURE CITED

It is now clear that all mature cells, including beta-cells, must be continually replaced and the number present depends not only on their birth rate, which reflects the frequency of cell division of the appropriate precursor cell, but also on the life span, which most likely reflects the timing of death by apoptosis (Mandrup-Poulsen, 2001). Changes in the rate of apoptosis of beta-cells accounts for previously unexplained diabetes related sympotoms. Moreover, attenuation of the rate of apoptosis of beta-cells may be a key mechanism for the effects of anti-diabetic agents, such as GLP-1. Proof of the principle that a cell population's function can be increased by suppression of apoptosis provides clues for the development of novel pharmacotherapeutic strategies for diabetic pathological conditions and islet transplantation in which tissue mass diminution has compromised functional integrity. Nevertheless, before we can definitively describe the pathogenesis and mechanism(s) of the various causes of beta-cell loss and thereby develop anti-diabetic drugs, there are areas that still require further in vivo studies; the beta-cell specific death pathway in each condition (Types I, II diabetes and islet transplantion), molecular markers used for determining the pancreatic apoptosis, as well as other mechanisms including changes in pancreas activity.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. APOPTOSIS IN PANCREATIC BETA-CELLS
  4. STIMULI OF APOPTOSIS IN PANCREATIC BETA-CELLS
  5. CASPASES: ACTIVATION AND PATHWAY
  6. FROM THE ACTIVATION OF CASPASES TO THE APOPTOTIC CHANGES IN THE MORPHOLOGY OF PANCREATIC BETA-CELLS
  7. STRATEGIES FOR PREVENTING APOPTOSIS OF ISLETS
  8. CONCLUSION
  9. Acknowledgements
  10. LITERATURE CITED
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