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PublisherThe University of Arizona.
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AbstractObesity rates have risen steeply in recent decades. This is accompanied with increased prevalence of several obesity-related disorders including type 2 diabetes. Genetic, environmental, and lifestyle factors (increased caloric intake and/or decreased physical activity) predispose individuals to type 2 diabetes by decreasing the body's responsiveness to the pancreatic hormone insulin, a physiological phenomenon commonly referred to as insulin resistance. Insulin resistance occurs due to induction of inflammation characterized by increased secretion of pro-inflammatory cytokines from enlarged adipose tissue, endoplasmic reticulum stress, and oxidative stress associated with excess blood glucose. There is a strong correlation between insulin resistance and decreased circulating levels of adiponectin (APN), a hormone implicated in promoting insulin-like activities. Further, inflammation negatively affects both insulin and adiponectin levels. It is recognized that folding of APN 18mer-subunits (insulin-sensitizing oligomer) is hindered in obese and type 2 diabetic individuals. Likewise, formation of normal hexameric insulin complex is compromised in type 2 diabetes. Insulin biogenesis, packaging, and assembly are impaired and unable to compensate for high blood glucose levels. As insulin and APN are key metabolic hormones essential for proper glucose regulation, maintaining their correct folding and assembly is required for conserving overall metabolic homeostasis. This dissertation centers on investigating proper assembly pathways of APN and insulin isoforms to form the higher order complexes necessary for their function. The interaction between APN oligomers was studied in the presence and absence of zinc, previously shown to inhibit formation of disulfide bonds in APN. We observed that zinc protects APN from collapse under acidic conditions and likely stabilizes oligomers through high affinity histidine coordination. The interaction between oligomers was further assessed by analyzing conformational differences between oligomers through tryptophan fluorescence. Reduced oligomers were observed to have significant structural differences compared to oxidized oligomers indicated by changes in fluorescent intensities. The capacity of APN chaperone DSBA-L to promote assembly was also evaluated although no significant changes were observed. In addition, the interaction between zinc and insulin was assessed where we observed that in the presence of zinc, insulin is significantly protected from reduction and precipitation. Zinc formed large complexes with insulin under reducing environments to induce high structural stability of insulin oligomers. We then utilized the strong conformational stability of insulin to develop a novel insulin analog with properties to slowly release insulin in circulation and more quickly in the presence of high glucose concentrations. Insulin modification is at preliminary stages and requires further experimentation. Together, these results indicate that zinc plays a significant role in multimerizing properties to provide high stability towards APN and insulin structures. Zinc enhances multimerization of oligomers to both promote activity of APN and protect insulin from reduction and premature breakdown to monomers. Through this study we better identified the folding pathway of APN and elucidated the strong intermolecular forces involved in oligomer association. In addition, the multimerization pattern of insulin to large conformation complexes is observed to mediate protection under reducing conditions. This has implications in the development of new therapeutic options to promote insulin-sensitization and insulin activity to regulate plasma glucose levels. In addition, we propose the development of a novel insulin-analog to mimic physiological insulin secretion, currently unavailable in the market.
Degree ProgramGraduate College
Molecular & Cellular Biology