The endoplasmic reticulum (ER) is the organelle responsible for synthesis and folding of secreted and membranous protein and lipid biosynthesis. It also functions as one of the main cellular calcium stores. Pancreatic beta-cells evolved to produce and secrete insulin upon demand in order to regulate blood glucose homeostasis. In response to increases in serum glucose, insulin synthesis represents nearly 50% of the total protein biosynthesis by beta-cells. This poses an enormous burden on the ER, rendering beta-cells vulnerable to agents that perturb ER function. Alterations of ER homeostasis lead to accumulation of misfolded proteins and activation of an adaptive response named the unfolded protein response (UPR). The UPR is transduced via 3 ER transmembrane proteins, namely PERK, IRE-1 and ATF6. The signaling cascades activated downstream of these proteins: a) induce expression of ER resident chaperones and protein foldases. Increasing the protein folding capacity of the ER; b) attenuate general protein translations which avoids overloading the stressed ER with new proteins; c) upregulate ER-associated degradation (ERAD) genes, which decreases the unfolded protein load of the ER. In severe cases, failure by the UPR to solve the ER stress leads to apoptosis. The mechanisms linking ER stress to apoptosis are still poorly understood, but potential mediators include the transcription factors Chop and ATF3, pro-apoptotic members of the Bcl-2 familly, the caspase 12 and the kinase JNK. <p>Accumulating evidence suggest that ER stress contributes to beta-cell apoptosis in both type 1 and type 2 diabetes. Type 1 diabetes is characterized by a severe insulin deficiency resulting from chronic and progressive destruction of pancreatic beta-cells by the immune system. During this autoimmune assault, beta-cells are exposed to cytokines secreted by the immune cells infiltrating the pancreatic islets. Our group has previously shown that the pro-inflamatory cytokines interleukin-1beta (IL1-beta and interferon-gamma (IFN-gamma), via nitric oxide (NO) formation, downregulate expression and function of the ER Ca2+ pump SERCA2. This depletes beta-cell ER Ca2+ stores, leading to ER stress and apoptosis. Of note, IL1-beta alone triggers ER stress but does not induce beta-cell death, while IFN-gamma neither causes ER stress nor induces beta-cell death. Together, these cytokines cause beta-cell apoptosis but the mechanisms behind this synergistic effect were unknown.<p>Type 2 diabetes is characterized by both peripheral resistance to insulin, usually as a result of obesity, and deficient insulin secretion secondary to beta cell failure. Obese patients have high levels of circulating free fatty acids (FFA) and several studies have shown that the FFA palmitate induces ER stress and beta-cell apoptosis.<p>In the present work we initially established an experimental model to specifically activate the ER stress response in pancreatic beta-cells. For this purpose, insulinoma cells (INS-1E) or primary rat beta-cells were exposed to the reversible chemical SERCA pump blocker cyclopiazonic acid (CPA). Dose-response and time course experiments determined the best conditions to induce a marked ER stress without excessive cell death (<25%).<p>The first goal of the work was to understand the synergistic effects of IL1-beta and IFN-gamma leading to pancreatic beta-cell apoptosis. Our group previously observed, by microarray analysis of primary beta-cells, that IFN-gamma down-regulates mRNAs encoding for some ER chaperones. Against this background, our hypothesis was that IFN-gamma aggravates beta-cell ER stress by decreasing the ability of these cells to mount an adequate UPR. To test this hypothesis, we investigated whether IFN-gamma pre-treatment augments CPA-induced ER stress and beta cell death. The results obtained indicated that IFN-gamma pre-treatment potentiates CPA-induced apoptosis in INS-1E and primary beta-cells. This effect was specific for IFN-gamma since neither IL1-beta nor a low dose CPA pre-treatment potentiated CPA-induced apoptosis in INS-1E cells. These effects of IFN-gamma were mediated via the down regulation of genes involved in beta cell defense against ER stress, including the ER chaperones BiP, Orp150 and Grp94 as well as Sec61, a component of the ERAD pathway. This had functional consequences as evidenced by a decreased basal and CPA-induced activity of a reporter construct for the unfolded protein response element (UPRE) and augmented expression of the pro-apoptotic transcription factor Chop. <p>We next investigated the molecular regulation of the Chop gene in INS-1E cells in response to several pro-apoptotic and ER stress inducing agents, namely cytokines (IL1-beta and IFN-gamma), palmitate, or CPA. Detailed mutagenesis studies of the Chop promoter showed differential regulation of Chop transcription by these compounds. While cytokines (via NO production)- and palmitate-induced Chop expression was mediated via a C/EBP-ATF composite and AP-1 binding sites, CPA induction required the C/EBP-ATF site and the ER stress response element (ERSE). Cytokines, palmitate and CPA induced ATF4 protein expression and further binding to the C/EBP-ATF composite site, as shown by Western blot and EMSA experiments. There was also formation of distinct AP-1 dimers and binding to the AP-1 site after exposure to cytokines or palmitate. <p>\ / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished
| Identifer | oai:union.ndltd.org:ulb.ac.be/oai:dipot.ulb.ac.be:2013/210623 |
| Date | 26 November 2007 |
| Creators | Pirot, Pierre |
| Contributors | Cardozo, Alessandra K, Eizirik, Decio, Lebrun, Philippe, Herchuelz, André, Heimberg, Harry, Vandenabeele, Peter, Robberecht, Patrick, Blanpain, Cédric |
| Publisher | Universite Libre de Bruxelles, Université libre de Bruxelles, Faculté de Médecine – Sciences biomédicales, Bruxelles |
| Source Sets | Université libre de Bruxelles |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:ulb-repo/semantics/doctoralThesis, info:ulb-repo/semantics/openurl/vlink-dissertation |
| Format | 1 v., No full-text files |
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