Superoxide anion imaging reveals physiological and pathological insights [electronic resource]

Reactive oxygen species (ROS) include a variety of highly reactive molecules that are produced during several normal biological processes. Amongst them, superoxide anion and hydrogen peroxide are some of the better studied. ROS are best known for their ability to cause oxidative stress, which can promote a variety of pathologies ranging from inflammation to cancer and aging. However, this limited view is changing. ROS are no longer thought of as unfortunate by-products of cellular respiration, but as crucial cell signaling molecules required for normal cell function and organismal survival. Researching the regulation and role of ROS in health and disease is complicated by their short half-lives and instability. My work aimed to expand the current understanding of the roles of ROS by first validating and then applying the chemiluminescent superoxide anion reporter coelenterazine to dynamically monitor superoxide anion concentrations in vitro and in vivo in response to a variety of physiological and pathological stimuli. ROS are tightly linked to glucose homeostasis both at the cellular and organism level. At the cellular level, oxidative phosphorylation is considered the major source of basal superoxide anion; additional sources include cell-signaling events mediated by a variety of ligands including inflammatory cytokines. At the organism level, ROS serve as signaling molecules in the actions of insulin secretion and response. As such, I begin in chapter 2 evaluating the broader impact of the immune system on insulin resistance. Recent research has demonstrated a significant role of the adaptive immune system in insulin resistance; however, its relevance in the physiological regulation of insulin sensitivity versus the pathological dysregulation induced by a high-fat diet is less clear. By application of a severe-combined immunodeficient mouse model, I show that the adaptive immune system has a fundamental role in the maintenance of physiological insulin sensitivity; in contrast, however, a high-fat diet can induce glucose intolerance independent of the adaptive immune system. Chapters 3, 4 and 5 focus on superoxide anion detection in vitro and in vivo in response to various physiological and pathological stimuli. In chapter 3, the use of coelenterazine for the detection of superoxide anion reveals the temporally and kinetically dynamic cellular response to glucose in vitro. In vivo, superoxide anion concentrations are greatest within the [beta] cells of the pancreas and serve as a biomarker for [beta]-cell mass, predicting susceptibility to diabetes mellitus in the non-obese diabetic mouse model. In chapter 4, high-throughput coelenterazine imaging showed phenotypically distinct superoxide anion concentrations amongst different cancer cell lines. Additionally, the by-product of the coelenterazine chemiluminescent reaction, coelenteramide, is demonstrated to be detectable by fluorescent microscopy and flow cytometry. Finally, coelenterazine revealed higher concentrations of superoxide anion at tumor sites. Chapter 5 focuses on superoxide anion in the context of inflammation. Coelenterazine detects the dynamic response of an oxidative burst in vitro. In vivo, coelenterazine imaging revealed shifts in superoxide anion foci prior to the development of clinical signs in a mouse model of inflammatory bowel disease. In chapter 6, I take a broader approach at evaluating the health or disease status of the entire organism. Blood samples collected from diseased mice, those treated with lipopolysaccharide, with asymptomatic inflammatory bowel disease, or with mammary adenocarcinoma, all produced significantly greater chemiluminescence in the presence of coelenterazine than control, healthy, blood samples. As such, coelenterazine may offer an efficient, cost-effective method for first-line detection of health versus disease, allowing improved patient screening and monitoring.

Keywords: Biology.
Advisor: Contag Christopher H. primary advisor. Thesis advisor
Jones Patricia
McLaughlin Tracey advisor. Thesis advisor