| 1. |
- Andersson, Annika K., 1974-
(author)
-
Role of Inducible Nitric Oxide Synthase and Melatonin in Regulation of β-cell Sensitivity to Cytokines
- 2003
-
Doctoral thesis (other academic/artistic)abstract
- The mechanisms of β-cell destruction leading to type 1 diabetes are complex and not yet fully understood, but infiltration of the islets of Langerhans by autoreactive immune cells is believed to be important. Activated macrophages and T-cells may then secrete cytokines and free radicals, which could selectively damage the β-cells. Among the cytokines, IL-1β, IFN-γ and TNF-α can induce expression of inducible nitric synthase (iNOS) and cyclooxygenase-2. Subsequent nitric oxide (NO) and prostaglandin E2 (PGE2) formation may impair islet function.In the present study, the ability of melatonin (an antioxidative and immunoregulatory hormone) to protect against β-cell damage induced by streptozotocin (STZ; a diabetogenic and free radical generating substance) or IL-1β exposure was examined. In vitro, melatonin counteracted STZ- but not IL-1β-induced islet suppression, indicating that the protective effect of melatonin is related to interference with free radical generation and DNA damage, rather than NO synthesis. In vivo, non-immune mediated diabetes induced by a single dose of STZ was prevented by melatonin.Furthermore, the effects of proinflammatory cytokines were examined in islets obtained from mice with a targeted deletion of the iNOS gene (iNOS -/- mice) and wild-type controls. The in vitro data obtained show that exposure to IL-1β or (IL-1β + IFN-γ) induce disturbances in the insulin secretory pathway, which were independent of NO or PGE2 production and cell death. Initially after addition, in particular IL-1β seems to be stimulatory for the insulin secretory machinery of iNOS –/- islets, whereas IL-1β acts inhibitory after a prolonged period. Separate experiments suggest that the stimulatory effect of IL-1β involves an increased gene expression of phospholipase D1a/b. In addition, the formation of new insulin molecules appears to be affected, since IL-1β and (IL-1β + IFN-γ) suppressed mRNA expression of both insulin convertase enzymes and insulin itself.
|
|
| 2. |
- Anvari, Ebrahim, et al.
(author)
-
The novel NADPH oxidase 4 inhibitor GLX351322 counteracts glucose intolerance in high-fat diet-treated C57BL/6 mice
- 2015
-
In: Free radical research. - : Informa UK Limited. - 1071-5762 .- 1029-2470. ; 49:11, s. 1308-1318
-
Journal article (peer-reviewed)abstract
- In type 2 diabetes, it has been proposed that pancreatic beta-cell dysfunction is promoted by oxidative stress caused by NADPH oxidase (NOX) overactivity. Five different NOX enzymes (NOX1-5) have been characterized, among which NOX1 and NOX2 have been proposed to negatively affect beta-cells, but the putative role of NOX4 in type 2 diabetes-associated beta-cell dysfunction and glucose intolerance is largely unknown. Therefore, we presently investigated the importance of NOX4 for high-fat diet or HFD-induced glucose intolerance using male C57BL/6 mice using the new NOX4 inhibitor GLX351322, which has relative NOX4 selectivity over NOX2. In HFD-treated male C57BL/6 mice a two-week treatment with GLX351322 counteracted non-fasting hyperglycemia and impaired glucose tolerance. This effect occurred without any change in peripheral insulin sensitivity. To ascertain that NOX4 also plays a role for the function of human beta-cells, we observed that glucose- and sodium palmitate-induced insulin release from human islets in vitro was increased in response to NOX4 inhibitors. In long-term experiments (1-3 days), high-glucose-induced human islet cell reactive oxygen species (ROS) production and death were prevented by GLX351322. We propose that while short-term NOX4-generated ROS production is a physiological requirement for beta-cell function, persistent NOX4 activity, for example, during conditions of high-fat feeding, promotes ROS-mediated beta-cell dysfunction. Thus, selective NOX inhibition may be a therapeutic strategy in type 2 diabetes.
|
|
| 3. |
- Barbu, Andreea Roxana, 1973-
(author)
-
In vitro Studies of β-cell Death and Survival. Modulation by Adenoviral Vectors and Bcl-2 Overexpression
- 2004
-
Doctoral thesis (other academic/artistic)abstract
- Type 1 diabetes is a multifactorial disease resulting from the selective destruction of insulin-producing β-cells within the pancreatic islets of Langerhans. The mechanisms of β-cell death are not fully understood but cytokines are important mediators of this process. In the present study we found that the combination of IL-1β, TNF-α and IFN-γ induced a nitric oxide-dependent disruption of the mitochondrial membrane potential in rat insulin-producing RINm5F-cells, which seems to be a necessary event for both RINm5F-cell apoptosis and necrosis. The antiapoptotic protein Bcl-2 was able to prevent cellular death in RINm5F cells, most probably by counteracting the mitochondrial permeability transition. These results pointed out the potential of such antiapoptotic genes as gene therapy tools, to allow enhanced resistance against autoimmune destruction of β-cells in type 1 diabetes. For this purpose we used a progesterone-antagonist (RU 486)-inducible gene transfer system to achieve an efficient and controlled Bcl-2 overexpression in primary rat β-cells. However, in our experience, prolonged in vitro culture revealed adenoviral-induced islet cell necrosis, a process that was not prevented by Bcl-2 overexpression. Moreover, we observed that specific adenoviral genotypes correlate with differential induction of necrosis in both human and rat pancreatic islet cells. Although human islet cells showed an increased resistance in terms of viral concentrations required for the induction of cell-toxicity, our results showed that they were unable to build up an efficient antiviral response following infection and that their survival was dependent on the exogenous addition of α-interferon.In conclusion, adenoviral techniques for overexpression of antiapoptotic proteins in insulin-producing cells may provide useful tools against β-cell directed autoimmune destruction. However, understanding the specific interactions of the viral gene products with cellular proteins and how they are involved in β-cell death regulation is fundamental for an efficient and safe application of gene therapy approaches to type 1 diabetes.
|
|
| 4. |
|
|
| 5. |
|
|
| 6. |
|
|
| 7. |
- Elksnis, Andris, 1993-
(author)
-
Pharmaceutical Protection of Beta-Cells in Diabetes : Using Tyrosine Kinase Inhibition and NOX4 Inhibitors
- 2022
-
Doctoral thesis (other academic/artistic)abstract
- Diabetes mellitus is a complex and heterogenous disease, with loss of beta-cell function and mass being a characteristic of not only type 1 diabetes (T1D), but also type 2 diabetes (T2D). In T1D, inappropriate inflammatory signaling is thought to participate in the autoimmune suppression and destruction of beta-cells. In T2D progressive insulin resistance with resulting glucolipotoxicity, increased inflammation and oxidative stress, drives islet amyloid formation and subsequent beta-cell exhaustion and failure. Even under best managed care, disease progression and eventual complications are unavoidable. New interventions that aim to improve beta-cell survival are highly needed. This thesis investigates two such possible interventions: the tyrosine kinase inhibitor Imatinib, and selective NADPH-oxidase inhibition.Imatinib mesylate, used in treatment of chronic myeloid leukemia and other malignancies, was soon after its introduction reported to possess anti-diabetic properties in both T1D and T2D patients undergoing treatment. Imatinib has been shown to prevent and reverse diabetes in NOD mice and improve glucose tolerance in high fat diet treated rats. In paper I, we aimed to characterize the mechanisms by which imatinib protects beta-cells. We found that imatinib inhibits complex I and II of the respiratory chain, leading to improved beta-cell survival through AMPK activation, reduced amyloid formation and protection against TXNIP upregulation.Oxidative stress may play a pivotal role in the development of beta-cell dysfunction and failure in T2D. The NADPH-oxidases are a family of 7 enzymes (NOX1-5 and DUOX 1-2), that produce reactive oxygen species that are important in various physiological processes but may, if excessively activated, also be a source for oxidative stress in T2D. In paper II, we evaluate novel selective NOX inhibitors as protective agents against in vitro induced human beta-cell stress. Selective NOX4 inhibition protected beta-cells against both cytokines and high-glucose + palmitate. In paper III we found that NOX4 inhibition increased mitochondrial membrane potential, mitochondrial reactive oxygen species and ATP/ADP ratio in a human beta-cell line, and this was paralleled with protection against human islet cell death when challenged with high-glucose and palmitate. Finally, in paper IV, we attempt to apply these findings in vivo, by transplanting athymic diabetic mice with human islets and treating them with a NOX4 inhibitor over a period of 4 weeks. Treated mice achieved lower blood glucose levels and water consumption throughout the treatment period, and apoptotic rates of insulin-positive human cells, measured as co-localization of insulin and cleaved caspase-3, were greatly reduced.
|
|
| 8. |
- Elksnis, Andris, et al.
(author)
-
Pharmacological Inhibition of NOX4 Improves Mitochondrial Function and Survival in Human Beta-Cells
- 2021
-
In: Biomedicines. - : MDPI. - 2227-9059. ; 9:12
-
Journal article (peer-reviewed)abstract
- Previous studies have reported beneficial effects of NADPH oxidase 4 (NOX4) inhibition on beta-cell survival in vitro and in vivo. The mechanisms by which NOX4 inhibition protects insulin producing cells are, however, not known. The aim of the present study was to investigate the effects of a pharmacological NOX4 inhibitor (GLX7013114) on human islet and EndoC-beta H1 cell mitochondrial function, and to correlate such effects with survival in islets of different size, activity, and glucose-stimulated insulin release responsiveness. We found that maximal oxygen consumption rates, but not the rates of acidification and proton leak, were increased in islets after acute NOX4 inhibition. In EndoC-beta H1 cells, NOX4 inhibition increased the mitochondrial membrane potential, as estimated by JC-1 fluorescence; mitochondrial reactive oxygen species (ROS) production, as estimated by MitoSOX fluorescence; and the ATP/ADP ratio, as assessed by a bioluminescent assay. Moreover, the insulin release from EndoC-beta H1 cells at a high glucose concentration increased with NOX4 inhibition. These findings were paralleled by NOX4 inhibition-induced protection against human islet cell death when challenged with high glucose and sodium palmitate. The NOX4 inhibitor protected equally well islets of different size, activity, and glucose responsiveness. We conclude that pharmacological alleviation of NOX4-induced inhibition of beta-cell mitochondria leads to increased, and not decreased, mitochondrial ROS, and this was associated with protection against cell death occurring in different types of heterogeneous islets. Thus, NOX4 inhibition or modulation may be a therapeutic strategy in type 2 diabetes that targets all types of islets.
|
|
| 9. |
- Elksnis, Andris, et al.
(author)
-
The selective NOX4 inhibitor GLX7013159 decreases blood glucose concentrations and human beta-cell apoptotic rates in diabetic NMRI nu/nu mice transplanted with human islets
- 2023
-
In: Free radical research. - : Taylor & Francis. - 1071-5762 .- 1029-2470. ; 57:6-12, s. 460-469
-
Journal article (peer-reviewed)abstract
- NADPH oxidase 4 (NOX4) inhibition has been reported to mitigate diabetes-induced beta-cell dysfunction and improve survival in vitro, as well as counteract high-fat diet-induced glucose intolerance in mice. We investigated the antidiabetic effects of the selective NOX4 inhibitor GLX7013159 in vivo in athymic diabetic mice transplanted with human islets over a period of 4 weeks. The GLX7013159-treated mice achieved lower blood glucose and water consumption throughout the treatment period. Furthermore, GLX7013159 treatment resulted in improved insulin and c-peptide levels, better insulin secretion capacity, as well as in greatly reduced apoptotic rates of the insulin-positive human cells, measured as colocalization of insulin and cleaved caspase-3. We conclude that the antidiabetic effects of NOX4 inhibition by GLX7013159 are observed also during a prolonged study period in vivo and are likely to be due to an improved survival and function of the human beta-cells.
|
|
| 10. |
- Fred, Rikard G.
(author)
-
The Role of RNA Binding Proteins in Insulin Messenger Stability and Translation
- 2010
-
Doctoral thesis (other academic/artistic)abstract
- Although the reason for insufficient release of insulin in diabetes mellitus may vary depending on the type and stage of the disease, it is of vital importance that an amplified insulin biosynthesis can meet the increased need during periods of hyperglycemia. The insulin mRNA is highly abundant in beta cells and changes in insulin mRNA levels are, at least in part, controlled by altered rates of mRNA degradation. Since the mechanisms behind the control of insulin messenger stability and translation are still largely obscure, the work presented in this thesis therefore aimed to further investigate the role of insulin mRNA binding proteins in the control of insulin mRNA break-down and utilization for insulin biosynthesis. To clarify how glucose regulates insulin mRNA stability and translation we studied the correlation between polypyrimidine tract binding protein (PTB) gene expression and insulin mRNA levels. It was found that an increase in PTB mRNA and protein levels is paralleled by an increase in insulin mRNA levels. It was also found that PTB binds to the 5’-untranslated region of the insulin mRNA and that insulin mRNA can be translated through a cap-independent mechanism in human islets of Langerhans, possibly due to the interaction with PTB. Further it was discovered that the suppressed insulin biosynthesis in human islets during glucotoxicity is partly due to an induction of the microRNA miR-133a. This induction leads to decreased levels of PTB and insulin biosynthesis rates in human islets. Finally, we were able to identify two proteins, hnRNP U and TIAR, that bind specifically to the insulin mRNA in vitro, and show that the stability and translation of insulin mRNA is oppositely affected by these proteins. In conclusion, insulin producing cells seem to be able to regulate insulin messenger stability and translation by a control mechanism in which the binding of specific proteins to the insulin messenger dictates the outcome. A better understanding of the events leading to insulin production may in the future aid in optimal diagnosis and treatment of type 2 diabetes.
|
|
