NF-κB and the pancreatic beta cell

As described above, type 1 diabetes mellitus is an auto-immune disease causing specific destruction of pancreatic beta cells. Pro-inflammatory cytokines, in particular IL-1β, IFN-γ and TNF-α, that are released by macrophages, monocytes and natural killer cells, play important roles in beta cell dysfunction and destruction associated with type 1 diabetes.

Activation of NF-κB by pro-inflammatory cytokines IL-1β and TNFα in the pancreatic beta cell is well documented, and this pathway is now considered as one of the major factors involved in beta cell dysfunction and death in type 1 diabetes (29; 32).

Increased production of nitric oxide (NO), due to induction of inducible nitric oxide synthase (iNOS), is thought to be an important signal in cytokine-induced cell death (29). IL-1β-induced iNOS expression is inhibited by treatment of cells with proteasome inhibitors that block NF-κB activation (diethyldithiocarbamate DETC, and PDTC) (275), suggesting that NF-κB plays a critical role in IL-1β-induced iNOS expression and in cytokine-induced beta cell death.

Adenoviral transfer of non-phosphorylatable and non-degradable IκBα variant (hence called Ad-IκB), that sequesters NF-κB in the cytoplasm, has also been used as a more specific means than proteasome inhibitors to assess the role of NF-κB in beta cells. Ad-IκB prevents IL-1β-induced dysfunction, NO production and Fas-triggered, IL-1β-mediated induction of apoptosis in human islets, indicating that NF-κB is involved in the deleterious effects of IL-1β leading to beta cell dysfunction and death (276). The protective effect of Ad-IκB against cytokine (IFNγ + IL-1β)-induced apoptosis has also been reported for purified rat beta cells (277). MIN6 clones that stably express the same IκB variant are protected against cytokine (IL-1β + IFNγ + TNFα)-induced apoptosis compared to control clones (278); and peptide-mediated transduction of the IKK inhibitor Nemo-binding domain (NBD), that inhibits IL-1β-induced NF-κB activation, blocks IL-1β-mediated dysfunction of mouse islets (279). Furthermore, in vivo transduction of this peptide in mice prior to isolation prevents loss of function and viability of islets due to isolation (279). Thus, these papers indicate that cytokine-induced NF-κB activity plays a pro-apoptotic role in pancreatic beta cells. However, there is one report that indicates that cytokine (TNFα)-induced NF-κB activity can have anti-apoptotic effects on the mouse insulinoma cell line MIN-6 and in primary mouse islets (280). Indeed, TNFα alone is not able to induce apoptosis of beta cells. However, treatment of cells with the proteasome inhibitor MG132 (that inhibits NF-κB) or with Ad-IκB sensitizes cells to TNFα-induced apoptosis, suggesting that TNFα-induced activation of NF-κB protects beta cells (MIN-6, mouse islets) from TNFα-mediated apoptosis (280).

To better understand the mechanisms of this NF-κB connection in cytokine-induced beta cell dysfunction and death, microarray analysis has been performed on purified primary rat beta cells that were treated or not with cytokines (IL-1β + IFNγ), and that were

infected with either a control adenovirus or with Ad-IκB (281). This approach led to the identification of 66 cytokine-modified and NF-κB-regulated genes in beta cells. Most notably, it was found that cytokine-induced NF-κB activation leads to an increase in the expression of pro-apoptotic genes (such as c-Myc, Fas and iNOS) and of chemokines and cytokines, that might contribute to the activation and the recruitment of inflammatory cells to the area of insulitis.

Furthermore, it was observed that cytokine-induced NF-κB activity leads to a decrease in the expression of genes involved in the maintenance of the differentiated beta cell functions, such as Pdx-1, Isl-1 and Glut-2. Finally, cytokine-induced modifications of the expression of several transcription factors, such as C/EPBβ, C/EBPδ, c-Myc and Isl-1 were found to be NF-κB-dependent (281). In summary, these data suggest that NF-κB is a key switch regulator of transcription factors and gene networks controlling cytokine-induced beta cell dysfunction and death occurring in the pathogenesis of type 1 diabetes.

Until recently, IL-1β production and release by islets was considered to be limited to type 1 diabetes. However, in a recent breakthrough report, it was suggested that this mechanism is also involved in the development of type 2 diabetes (282). As described above (Part I, § 1.2.2.), glucotoxicity is considered as one of the main mechanisms leading to apoptosis in type 2 diabetes. Maedler and colleagues have reported that culture of human islets with high glucose concentrations induces secretion of IL-1β by the beta cells themselves, leading to their dysfunction as well as an increase in beta cell death (282). Inhibition of IL-1β signaling (using IL-1Ra) blocks high glucose- and IL-1β-induced NF-κB activity; and blockade of IL-1β signaling and of NF-κB activity (with the proteasome inhibitor PDTC) block the deleterious effects of high glucose concentrations on both beta cell function and survival. Thus, these results indicate that release of IL-1β by beta cells cultured in high glucose concentrations is followed by NF-κB activation and, ultimately, by impaired beta cell function and survival (282). In addition, IL-1β-producing cells are observed in pancreatic sections of poorly controlled type 2 diabetic patients but not in non-diabetic control subjects (282). The diabetes-prone gerbil Psammomys obesus is an animal that develops type 2 diabetes upon high-energy diet (21).

Interestingly, IL-1β expression is induced in the beta cells of the diabetes-prone Psammomys obesus fed with a high-energy diet, but not in the diabetes-resistant counterparts (282). These

gene expression (230; 284-286). This seems to also be the case for the pancreatic beta cell.

Indeed, in the studies described above, beta cells were exposed for prolonged times to high doses of cytokines. However, it has been reported that when beta cells are exposed for a limited period (24h) to low concentrations of IL-1β, the protective effects of NF-κB-regulated genes preponderate and beta cells are protected against conditions that cause necrosis (287). This suggests that the effects of IL-1β-induced NF-κB activity are dose- and/or duration-dependent.

This concept is supported by a recent report indicating that the first phase of IL-1β-induced NF-κB activity leads to an increase of beneficial beta cell defense/repair protein expression (288).

By contrast, the second phase of NF-κB activity induced by IL-1β seems to be harmful because it then leads to a sustained decrease of specific beta cell proteins like insulin, Glut-2 and Pdx-1 with a concomitant increase of other “aspecific” proteins and iNOS transcription (288). However, in this study, the effects of IL-1β-induced NF-κB activity were assessed using the protease inhibitor TPCK, and thus remain to be confirmed with more specific means to inhibit NF-κB.

Other stimuli than cytokines have been shown to activate NF-κB in pancreatic beta cells, such as depolarization and Ca²⁺ influx (289). However, the consequences of this NF-κB activity remain to be characterized. GLP-1 has been reported to induce the NF-κB DNA-binding activity and the expression of the anti-apoptotic NF-κB target genes IAP-1 and Bcl-2 in INS-1 cells (190). Interestingly, GLP-1-induced protection of beta cells from glucolipotoxicity is abolished upon treatment of cells with the NF-κB inhibitor Bay 11-7082, suggesting that NF-κB mediates the anti-apoptotic effect of GLP-1 on beta cells cultured in glucolipotoxic conditions (190). Stable overexpression of c-FLIP in the mouse beta cell line βTc-Tet leads to protection of mouse beta cells against cytokine-induced caspase-8 activation and apoptosis, as well as to an enhanced NF-κB activity (290). Although this report does not show evidence that these two phenomena are related, it is proposed by the authors that protection of beta cells against cytokine-induced apoptosis by c-FLIP overexpression may be linked to its effect on NF-κB activity (290). Finally, in a very recent report Norlin and colleagues report the generation of mice harbouring beta-cell specific overexpression of a non-phosphorylatable mutant of IκBα that inhibits NF-κB activity (291). Interestingly, glucose-stimulated insulin secretion was impaired in the transgenic mice, while beta cell survival was not affected (291).

In summary, the majority of studies assessing the effects of IL-1β-induced NF-κB activity report a deleterious effect of this activity for pancreatic beta cell function and survival.

Furthermore, this signaling module has been suggested to be potentially involved in the pathogenesis of both types of diabetes. Little is known about the functional consequences of NF-κB activity induced by stimuli other than cytokines. However, these data suggest the functional consequences of NF-κB activity induced by stimuli other than cytokines may differ considerably from the deleterious effects of cytokine-induced NF-κB activity on the beta cell.

Finally, no studies on the possible effects of ECM and/or integrins on NF-κB activity have been reported yet for the pancreatic beta cell.