The islets of Langerhans secrets insulin in response to fluctuations of blood glucose level and efficient secretion requires extensive intra-islet communication. Secretory failure from islets is one of the hallmark in progression of type 2 diabetes.  Changes in islet structure and high levels of saturated free fatty acids may contribute to this failure. The aim of this thesis is to study the role of cell-cell interactions and palmitate on β-cells functions. To address the role of cell-cell interactions on β-cells functions MIN6 cells were cultured as monolayers and as pseudoislets. Glucose stimulated insulin secretion was higher in pseudoislets compared to monolayers. Transcript levels of mitochondrial metabolism as well glucose oxidation rate was higher in pseudoislets. Insulin receptor substrate-1 (IRS-1) phosphorylation was altered when cells were grown as pseudoislets. Proteins expression levels related to glycolysis, cellular connections and translational regulations were up-regulated in pseudoislets. We propose the superior capacity of pseudoislets compared to monolayers depend on metabolism, cell coupling, gene translation, protein turnover and differential IRS-1 phosphorylation. To address the role of palmitate on β-cells human islets were cultured in palmitate. Long term palmitate treatment decreased insulin secretion which is associated with up-regulation of suppressor of cytokine signaling-2 (SOCS2) and protein inhibitor of activated STAT-1 (PIAS1). Up-regulation of SOCS2 decreased phosphorylation of Akt at site T308, whereas PIAS1 decreased protein level of ATP- citrate lyase (ACLY) and ATP synthase subunit B (ATP5B). We propose long term palmitate treatment reduces phosphatidylinositol 3-kinase (PI3K) activity, attenuates formation of acetyl-CoA and decreases ATP synthesis which may aggravate β-cells dysfunction.

<p>Transplanting pancreatic islets into the liver through the portal vein is currently the most common procedure in clinical islet transplantations for treating patients with brittle type 1 diabetes. However, most islet grafts fail within a 5-year period necessitating retransplantation. The vascular connections are disrupted at islet isolation and implanted islets depend on diffusion of oxygen and nutrients in the immediate posttransplantation period. Rapid and efficient revascularization is of utmost importance for the survival and long-term function of transplanted islets. </p><p>In this thesis, the influence of the implantation microenvironment for islet engraftment and function was studied. Islets were transplanted into the liver, the renal subcapsular site or the pancreas. Islets implanted into the liver contained fewer glucagon-positive cells than islets implanted to the kidney and endogenous islets. Intraportally transplanted islets responded with insulin and glucagon release to secretagogues, but only when stimulated through the hepatic artery. Thus, the intrahepatic grafts were selectively revascularized from the hepatic artery. The vascular density in human islets transplanted into the liver of athymic mice was markedly lower when compared to human islets grafted to the kidney. Islets implanted into their physiological environment, the pancreas, were markedly better revascularized. Insulin content, glucose-stimulated insulin release, (pro)insulin biosynthesis and glucose oxidation rate were markedly decreased in transplanted islets retrieved from the liver, both when compared to endogenous and transplanted islets retrieved from the pancreas. Only minor changes in metabolic functions were observed in islets implanted into the pancreas when compared to endogenous islets. </p><p>The present findings demonstrate that the microenvironment has a major impact on the engraftment of transplanted islets. Elucidating the beneficial factors that promote engraftment would improve the survival and long-term function of transplanted islets. Ultimately, islet transplantation may be provided to an increased number of patients with type 1 diabetes.</p>

Transplanting pancreatic islets into the liver through the portal vein is currently the most common procedure in clinical islet transplantations for treating patients with brittle type 1 diabetes. However, most islet grafts fail within a 5-year period necessitating retransplantation. The vascular connections are disrupted at islet isolation and implanted islets depend on diffusion of oxygen and nutrients in the immediate posttransplantation period. Rapid and efficient revascularization is of utmost importance for the survival and long-term function of transplanted islets. In this thesis, the influence of the implantation microenvironment for islet engraftment and function was studied. Islets were transplanted into the liver, the renal subcapsular site or the pancreas. Islets implanted into the liver contained fewer glucagon-positive cells than islets implanted to the kidney and endogenous islets. Intraportally transplanted islets responded with insulin and glucagon release to secretagogues, but only when stimulated through the hepatic artery. Thus, the intrahepatic grafts were selectively revascularized from the hepatic artery. The vascular density in human islets transplanted into the liver of athymic mice was markedly lower when compared to human islets grafted to the kidney. Islets implanted into their physiological environment, the pancreas, were markedly better revascularized. Insulin content, glucose-stimulated insulin release, (pro)insulin biosynthesis and glucose oxidation rate were markedly decreased in transplanted islets retrieved from the liver, both when compared to endogenous and transplanted islets retrieved from the pancreas. Only minor changes in metabolic functions were observed in islets implanted into the pancreas when compared to endogenous islets. The present findings demonstrate that the microenvironment has a major impact on the engraftment of transplanted islets. Elucidating the beneficial factors that promote engraftment would improve the survival and long-term function of transplanted islets. Ultimately, islet transplantation may be provided to an increased number of patients with type 1 diabetes.

Type 1 diabetes is an autoimmune disease resulting in the selective destruction of the insulin producing β-cells in the pancreas. Pro-inflammatory cytokines and the free radical nitric oxide (NO) have been implicated in mediating the destruction of β-cells, possibly through activation of the mitogen activated protein kinases (MAPKs) JNK, ERK and p38. In addition to MAPKs, cytokine signaling also results in activation of the transcription factor nuclear factor-kappaB (NF-κB). The upstream signaling events leading to MAPK and NF-κB activation in β-cells are not well known. The work presented in this thesis therefore aims at characterizing the regulation of MAPKs and NF-κB in human islets, with emphasis on the role of the MAPK activator MAP/ERK kinase kinase-1 (MEKK-1) in islet cell death. It was found that MEKK-1 was phosphorylated in response to the nitric oxide donor DETA/NONOate (DETA/NO), the β-cell toxin streptozotocin (STZ) and pro-inflammatory cytokines and that MEKK-1 downstream signaling in response to the same treatments involved activation of JNK but not ERK and p38. MEKK-1 was also found to be essential for cytokine-induced NF-κB activation. MEKK-1 downregulation protected human islet cells from DETA/NO-, STZ, and cytokine-induced cell death. Furthermore, overexpression of the NF-κB subunit c-Rel protected human islet cells from STZ and hydrogen peroxide-induced cell death indicating that NF-κB activity protects against cell death in human islets. In summary, these results support an essential role for MEKK-1 in the activation of JNK and NF-κB, with important consequences for human islet cell death and that strategies preventing human islets death by inhibition of the JNK pathway instead of NF-κB might be suitable.

Chronic hyperlipidemia (lipotoxicity) and hyperglycemia (glucotoxicity) have recently been shown to induce Endoplasmic Reticulum (ER) stress, which may contribute to pancreatic beta-cell dysfunction in type 2 diabetes. This thesis examined the involvement of ER stress in beta-cell lipotoxicity and glucotoxicity. Although chronic treatment with saturated free fatty acids (FFA) in vitro induced ER stress, altering ER stress by increasing or knocking-down GRP78 chaperone expression had no effect on apoptosis induction. Conversely, overexpression of ER chaperones rescued the reduction in proinsulin protein levels caused by chronic exposure to high glucose, although it had no effect on the decreased insulin mRNA levels and proinsulin translation rate. Thus, ER stress is likely not the main mechanism involved in saturated FFA-induced beta-cell apoptosis in vitro, but it may contribute to glucotoxic effects on proinsulin levels. These findings have increased our understanding of the link between ER stress and beta-cell dysfunction in type 2 diabetes.

Chronic hyperlipidemia (lipotoxicity) and hyperglycemia (glucotoxicity) have recently been shown to induce Endoplasmic Reticulum (ER) stress, which may contribute to pancreatic beta-cell dysfunction in type 2 diabetes. This thesis examined the involvement of ER stress in beta-cell lipotoxicity and glucotoxicity. Although chronic treatment with saturated free fatty acids (FFA) in vitro induced ER stress, altering ER stress by increasing or knocking-down GRP78 chaperone expression had no effect on apoptosis induction. Conversely, overexpression of ER chaperones rescued the reduction in proinsulin protein levels caused by chronic exposure to high glucose, although it had no effect on the decreased insulin mRNA levels and proinsulin translation rate. Thus, ER stress is likely not the main mechanism involved in saturated FFA-induced beta-cell apoptosis in vitro, but it may contribute to glucotoxic effects on proinsulin levels. These findings have increased our understanding of the link between ER stress and beta-cell dysfunction in type 2 diabetes.