An important mechanism involved in the pathogenesis of type 2 diabetes is elevation of plasma free fatty acids which induce insulin resistance and may impair both β-cell function and mass (β-cell lipotoxicity). The objective of my thesis was to investigate the role of oxidative stress in β-cell lipotoxicity, using in vivo, ex vivo, and in vitro models. I used in vivo models of 48h i.v. oleate or olive oil infusion in Wistar rats followed by hyperglycemic clamps, or islet secretion studies ex vivo, and in vitro models of 48h exposure to oleate in isolated islets. My first study showed that 48h oleate infusion decreased the insulin response to a hyperglycemic clamp, an effect prevented by coinfusion of the antioxidants N-acetylcysteine and taurine. Similar to the findings in vivo, 48h infusion of oleate decreased glucose stimulated insulin secretion (GSIS) ex vivo, and induced oxidative stress in isolated islets, effects prevented by coinfusion of the antioxidants N-acetylcysteine, taurine, or tempol. Islets exposed to oleate or palmitate showed a decreased insulin response to high glucose and increased levels of oxidative stress, effects prevented by taurine. Therefore, my data are the first demonstration that oxidative stress plays a role in the decrease in β-cell secretory function induced by prolonged exposure to FFA, in vitro and in vivo. My second study addressed downstream effects of oxidative stress involving inflammation. A 48h infusion of oleate or olive oil decreased β-cell function during a hyperglycemic clamp, an effect prevented by coinfusion of the IKKβ inhibitor salicylate. GSIS in isolated islets was impaired by olive oil or oleate and restored by salicylate. These results suggest a potential role for both oxidative stress and inflammation in lipid-induced β-cell dysfunction. My third study addressed downstream effects of oxidative stress involving β-cell insulin signalling. A 48h infusion of oleate or olive oil decreased β-cell function during a hyperglycemic clamp, an effect prevented by coinfusion of the tyrosine phosphatase inhibitor bisperoxovanadate. GSIS in isolated islets was impaired by olive oil or oleate and restored by bisperoxovanadate, suggesting a role of FFA in decreasing β-cell function by induction of β-cell insulin resistance.

Chronic hyperglycemia, a hallmark of type 2 diabetes, can decrease β-cell function and mass (β-cell glucotoxicity); however, the mechanisms are incompletely understood. The objective was to examine the mechanisms of β-cell glucotoxicity using in vivo and ex vivo models. The hypothesis is that oxidative stress plays a causal role in high glucose-induced β-cell dysfunction in vivo via pathways that involve endoplasmic reticulum (ER) stress and JNK. The model of β-cell glucotoxicity was achieved by prolonged i.v. glucose infusion (to achieve hyperglycemia). In Study 1, 48h glucose infusion increased total and mitochondrial superoxide levels in islets, and impaired β-cell function in vivo and ex vivo. Co-infusion of the superoxide dismutase mimetic Tempol decreased total and mitochondrial superoxide, and prevented high glucose-induced β-cell dysfunction in vivo and ex vivo. These results suggest that increased superoxide generation plays a role in β-cell glucotoxicity. In Study 2, 48h glucose infusion increased activation of the unfolded protein response (XBP-1 mRNA splicing and phospho-eIF2α levels). This was partially prevented by Tempol. Co-infusion of the chemical chaperone 4-phenylbutyrate with glucose decreased spliced XBP-1 levels, and prevented high glucose-induced β-cell dysfunction in vivo and ex vivo. Co-infusion of 4-phenylbutyrate also decreased total and mitochondrial superoxide induced by high glucose. These results suggest that 1) ER stress plays a causal role in high glucose-induced β-cell dysfunction, and 2) there is a link between oxidative stress and ER stress in high glucose-induced β-cell dysfunction in vivo. In Study 3, JNK inhibition using the inhibitor SP600125 in rats or JNK-1 null mice prevented high glucose-induced β-cell dysfunction ex vivo and in vivo. SP600125 prevented high-glucose-induced β-cell dysfunction without decreasing total and mitochondrial superoxide levels. Both Tempol and 4-phenylbutyrate prevented JNK activation induced by high glucose. These results suggest a role of JNK activation in high glucose-induced β-cell dysfunction downstream of increased superoxide generation and ER stress in vivo. Together, the results suggest that 1) oxidative stress, ER stress and JNK activation are causally involved in β-cell glucotoxicity, and 2) High glucose-induced oxidative stress and ER stress are linked, and both impair β-cell dysfunction via JNK activation in vivo.

Smoking and environmental tobacco smoke (ETS) exposure are major risk factors for cardiovascular disease (CVD), although the exact components and pathophysiological mechanisms responsible for this association remain unclear. Polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (BaP), are ubiquitous environmental contaminants that form during organic material combustion and are thus found in cigarette smoke, vehicle exhaust particles, and air pollution. We hypothesize that PAHs are key agents responsible for mediating the cigarette smoke effects in the cardiovascular system, including increased oxidative stress, inflammation, and arterial stiffness.<p> Arterial stiffness is a powerful, independent predictor of cardiovascular risk and is regulated, in part, by vasoactive mediators derived from the endothelium. The first objective of this project was to determine whether pulse wave dP/dt collected from radiotelemetry-implanted rats is a reliable indicator of changes in arterial stiffness following administration of vasoactive drugs or acute ETS exposure. Anaesthetized rats were administered a single dose of saline (vehicle control), acetylcholine, norepinephrine, and N(G)-nitro-L-arginine methyl ester (L-NAME) via the tail vein, allowing a washout period between injections. Acetylcholine decreased and norepinephrine increased dP/dt compared to saline vehicle. Injection of the nitric oxide (NO) synthase inhibitor L-NAME decreased plasma nitrate/nitrite (NOx), but transiently increased dP/dt. For the ETS experiment, rats were exposed for one hour to sham, low dose ETS, or high dose ETS. Exposure to ETS did not significantly alter dP/dt or plasma endothelin-1 (ET-1) levels, but increased plasma NOx levels at the high ETS exposure and increased plasma nitrotyrosine levels in both ETS groups. In conclusion, acute changes in NO production via acetylcholine or L-NAME alter the arterial pulse wave dP/dt consistently with the predicted changes in arterial stiffness. Although acute ETS appears to biologically inactivate NO, a concomitant increase in NO production at the high ETS exposure may explain why ETS did not acutely alter dP/dt.<p> The second objective of this project was to compare the effects of subchronic ETS and BaP exposure on circadian blood pressure patterns, arterial stiffness, and possible sources of oxidative stress in radiotelemetry-implanted rats. Pulse wave dP/dt was used as an indicator of arterial stiffness, and was compared to both structural (wall thickness) and functional (NO production and bioactivity, ET-1 levels) features of the arterial wall. In addition, histology of lung, heart, and liver were examined as well as pulmonary and hepatic detoxifying enzyme activity (cytochrome P450 specifically CYP1A1). Daily ETS exposure for 28 days altered the circadian pattern of heart rate and blood pressure in rats, with a loss in the normal dipping pattern of blood pressure during sleep. Subchronic ETS exposure also increased dP/dt in the absence of any structural modifications in the arterial wall. Although NO production and ET-1 levels were not altered by ETS, there was increased biological inactivation of NO via peroxynitrite production (as indicated by increased plasma nitrotyrosine levels). Thus, vascular stiffness and failure of blood pressure to dip precede structural changes in rats exposed to ETS for 28 days. Exposure to ETS also caused increased number of lung neutrophils as well as increased CYP1A1 activity in lung microsomes.<p> Since ETS-induced increases in arterial stiffness occurred as early as day 7, radiotelemetry-implanted rats were exposed daily to intranasal BaP for 7 days. Similar to ETS, BaP exposure altered circadian blood pressure patterns and reduced blood pressure dipping during sleep. Thus, in support of part of our hypothesis, the PAH component of cigarette smoke may be responsible for the ETS-induced increase in blood pressure and the loss of dipping pattern during sleep. Increased neutrophil recruitment was observed in the lungs of both ETS- and BaP-exposed rats, suggesting that lung inflammatory reactions may be involved in the disruption of circadian blood pressure rhythms. Unlike ETS however, BaP exposure did not significantly alter pulse wave dP/dt, endothelial function, or lung CYP1A1 activity. Thus, contrary to our hypothesis, the reduction in NO bioactivity and increased arterial stiffness caused by ETS cannot be explained by BaP at the dose and length of the exposure in the current study. Production of reactive metabolites in the lung following ETS exposure may be responsible, at least in part, for the increases in oxidative stress in the vasculature, leading to reduced NO bioactivity and increased arterial stiffness. Oxidative stress caused by BaP exposure may have been insufficient to reduce NO bioactivity in the peripheral vasculature. Therefore arterial stiffness was not increased and factors other than NO may be responsible for the increase in blood pressure observed with ETS and BaP exposure.

Uncoupling protein-2 (UCP2) may be linked to the attenuation of reactive oxygen species (ROS), but it is unclear whether this phenomenon pertains to the pancreatic beta cell. In this study, a UCP2-deficient mouse model was used to assess the importance of UCP2 to beta cell viability. We investigated the effect of UCP2 absence in response to a beta cell cytotoxic model of diabetes induction. In vivo treatment by the cytotoxic agent streptozotocin led to overall beta cell loss, but severity was not exacerbated by UCP2 deficiency. We also examined ROS production and cell viability in islet cells exposed to various stressors associated with oxidative stress. In vitro measurements of ROS and cell death in islet cells demonstrated that the response was not influenced by UCP2 expression. In contrast with UCP2 overexpression studies showing cytoprotection, this study reveals that beta cell survival is not compromised by the absence of UCP2.

Subarachnoid hemorrhage (SAH) comprises 7% of all stroke cases, and is associated with a disproportionately high morbidity and mortality with few therapeutic options available. The goal of this project was to understand the mechanism of neurological deterioration after experimental SAH, with a focus on cerebral vasospasm and brain injury after SAH. We tested the hypothesis that endothelial nitric oxide synthase (eNOS) is upregulated and uncoupled after SA, resulting in exacerbated neurological injury in a mouse model of SAH. The project entailed the investigation of eNOS-dimer uncoupling, its association with oxidative and nitrosative stress in the brain parenchyma and finally its association with secondary complications after SAH. In our studies we demonstrated the crucial role eNOS plays in anti-microthromboembolism, anti-apoptosis and maintenance of physiological superoxide (O2-)/NO balance. This study suggests that SAH up-regulates and disrupts eNOS, producing peroxynitrite (OONO-) and other radicals that further exacerbate the oxidative insult and neurological injury.

Oxidative stress has been implied in a wide variety of diseases, such as cancer, myocardial infarction, and neurodegenerative diseases including Parkinson's diseases (PD). PD is characterized by the degeneration of dopaminergic (DA) neurons; genetic studies have identified gene mutations causal to PD. Accumulating studies hypothesize that these genes protect DA neurons against oxidative stress. However, lack of experimental tools to target oxidative stress in specific cells has prevented direct evaluation of the hypothesis. We established a novel method to use KillerRed (KR), a genetically-encoded protein that generates radicals upon light activation. We showed its efficacy in live animals by cell-specific ablation of neurons in C. elegans. We applied KR to degenerate DA neurons. By controlling the level of stress via activation light, the protective role of PD-gene, LRRK2, against oxidative stress was confirmed. Thus, we established a method to address the role of oxidative stress in a cell-specific manner.

Subarachnoid hemorrhage (SAH) comprises 7% of all stroke cases, and is associated with a disproportionately high morbidity and mortality with few therapeutic options available. The goal of this project was to understand the mechanism of neurological deterioration after experimental SAH, with a focus on cerebral vasospasm and brain injury after SAH. We tested the hypothesis that endothelial nitric oxide synthase (eNOS) is upregulated and uncoupled after SA, resulting in exacerbated neurological injury in a mouse model of SAH. The project entailed the investigation of eNOS-dimer uncoupling, its association with oxidative and nitrosative stress in the brain parenchyma and finally its association with secondary complications after SAH. In our studies we demonstrated the crucial role eNOS plays in anti-microthromboembolism, anti-apoptosis and maintenance of physiological superoxide (O2-)/NO balance. This study suggests that SAH up-regulates and disrupts eNOS, producing peroxynitrite (OONO-) and other radicals that further exacerbate the oxidative insult and neurological injury.

Oxidative stress has been implied in a wide variety of diseases, such as cancer, myocardial infarction, and neurodegenerative diseases including Parkinson's diseases (PD). PD is characterized by the degeneration of dopaminergic (DA) neurons; genetic studies have identified gene mutations causal to PD. Accumulating studies hypothesize that these genes protect DA neurons against oxidative stress. However, lack of experimental tools to target oxidative stress in specific cells has prevented direct evaluation of the hypothesis. We established a novel method to use KillerRed (KR), a genetically-encoded protein that generates radicals upon light activation. We showed its efficacy in live animals by cell-specific ablation of neurons in C. elegans. We applied KR to degenerate DA neurons. By controlling the level of stress via activation light, the protective role of PD-gene, LRRK2, against oxidative stress was confirmed. Thus, we established a method to address the role of oxidative stress in a cell-specific manner.

Metal toxicity is implicated in neurotoxicity, nephrotoxicity, aging and cancer. Protein oxidation resulting from oxidative stress is now known to be involved in metal toxicity. However, proteomic responses to metal induced oxidative stress have not been characterized. By using the yeast as a model, we characterized these changes occurring in response to sub-lethal doses of metals. Several proteins involved in protein synthesis, ribosome assembly decreased while antioxidant defenses, proteins involved in sulfur metabolism, and glutathione synthesis and ubiquitin increased following metal exposure. We also show that metals induced temporal and targeted protein oxidation independent of protein abundance. Among the targets were glycolytic enzymes and heat-shock proteins. As a consequence, glycolytic enzyme activities decreased whereas the levels and activities of the enzymes of the alternative pathway for glucose metabolism, pentose phosphate pathway (PPP) increased. True to prediction, we also found increased flow through the PPP as measured by elevated levels of NADPH and glutathione. NADPH and glutathione are crucial for maintaining the redox balance in the cell. Thus, rerouting of glucose metabolism into PPP is considered to be beneficial to the organism. Among the oxidation targets is a glycolytic protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that is required for apoptosis in neuronal cells. We show that not only is GAPDH required for metal induced apoptosis in yeast but also the levels of GAPDH transcript and protein increase in the cytosol and the nucleus in an isoform specific fashion. Such changes strongly implicate the role of GAPDH in yeast apoptosis. This work provides evidence for the involvement of targeted protein oxidation in metal toxicity, shows the overlaps and differences in the mechanism of copper and cadmium toxicity, allows comprehension of how metabolic processes respond to metal stress and explores the potential of GAPDH as a sensor of oxidative stress and mediator for apoptosis.

Oxidative stress has been associated with many disease conditions in adults and neonates based on clinical and post mortem studies. Trauma is the commonest cause of oxidative stress. However a gap in knowledge of the natural history of oxidative stress in humans was identified as most studies have been post mortem or in animals. <p>The aim of this research is to understand treat and oxidative stress in trauma and surgical patients. The study involved three components including: the development and evaluation of the novel oxistress assay; study of clinical trauma and oxidative stress; and clinical trial of alanyl-glutamine supplementation following major surgery. The novel oxistress assay was used on urine samples in the normal population to determine reference values and subsequently on hospital patients to determine sensitivity and specificity. The study of clinical trauma and oxidative stress evaluated plasma antioxidants (FRAP assay), red cell glutathione (Asensis method), plasma and urine protein carbonyl (Levines method) and total oxidants in plasma and urine (oxistress assay) over 7 day period following trauma. The clinical trial was a double blind study of 69 major surgery patients evaluating biochemical and clinical parameters over 7 day period in comparison with pre-operative status. <p>The novel oxistress assay proves to be a sensitive and accurate bedside diagnostic tool for oxidative stress. It can also be used in the laboratory setting. Oxidative stress is associated with increased trauma severity resulting in antioxidant depletion, strong oxidant production and protein degradation. The presence of pre-morbid medical factors also increased oxidative stress in trauma patients. Oral alanyl-glutamine supplementation (0.3 g/kg) increased plasma glutamine and antioxidant levels while decreasing urine oxidant levels. It significantly reduced hospital stay in non-cancer and higher disease complexity patients. The intervention also reduced the resource intensity weighting (RIW) score. <p>Oxidative stress is a clinical problem in surgery and trauma patients that can now be easily diagnosed at the bedside using the novel oxistress assay. Treatment with alanyl-glutamine is effective in reducing oxidative stress and improving clinical outcome. It is highly recommended probably at a higher dose in order to achieve optimal results.