Murray State Theses and Dissertations

Abstract

Blood glucose homeostasis is a critical component in the physiological health of vertebrates. Regulation of blood glucose levels is the responsibility of the endocrine pancreas, which excretes hormones to the bloodstream in response to changes in glucose concentrations. The hormones excreted from the pancreas include insulin, which is secreted form the β cells of the pancreas and signals uptake of excess glucose by the peripheral tissues, and glucagon, which is secreted from the α cells and signals release of glucose to the bloodstream through gluconeogenesis in the liver. The Krüppel-like zinc finger protein Gli-Similar 3 (Glis3) is a transcription factor that has been shown to play a critical role in the development of β cells and transcription of the insulin gene.

Defects in the secretion and production of insulin are associated with the development metabolic diseases such as diabetes mellitus. Using genome wide association studies, GLIS3 has been identified as a risk locus for the development of both type 1 and type 2 diabetes mellitus and neonatal diabetes and hypothyroidism (NDH). Mice with ubiquitous knockouts of Glis3 exhibit phenotypes similar to that of humans, but conditional knockouts specific to the pancreas have produced mice with less severe phenotypes, leaving the role of Glis3 largely enigmatic.

The zebrafish (Danio rerio) has emerged as a powerful organism in studying pancreatic development due to its rapid development, short generation time, and transparent, externally developing embryos. The zebrafish pancreas is both morphologically and functionally comparable to that of humans, and the developmental programs that are responsible for pancreatic development appear to be highly conserved between the two. Zebrafish also have the unique ability to regenerate their β cells after complete ablation without the need for insulin treatment. These factors have made zebrafish a powerful model for the study of pancreatic development and diabetes.

We found that expression of glis3 mRNA begins after 14.5 hours post fertilization (hpf) in zebrafish. Zebrafish pancreas formation begins at approximately 24hpf, but whole mount in situ hybridization data shows that glis3 expression is restricted to the zebrafish brain until approximately 48hpf, when expression can be visualized in the pancreas. During this time frame glis3 expression levels increase, which coincide with the visualization of glis3 in the zebrafish pancreata.

Finally, we found that reduction of glis3 through a knockout mutation led to increased production of insulin and glucagon mRNA. Additionally, glis3 heterozygous mutants exhibited a heightened ability to control blood glucose levels, with less variability of blood glucose levels between individuals under fasting, postprandial, and high fat diet conditions when compared to wildtype fish. After four months exposure to a high fat, high glucose diet, wildtype zebrafish expressed heightened levels of resting blood glucose, while heterozygous mutants showed no increase in blood glucose levels when compared to a normal diet.

Collectively, the results of this project provide novel information regarding the regulation of insulin in glis3 mutant zebrafish. A decrease in glis3 expression levels may increase the propensity for an organism to begin the compensatory mechanism for β cell mass expansion, giving insight into possible models for clinical therapies for the treatment of diabetes.

Year manuscript completed

2018

Year degree awarded

2018

Author's Keywords

Diabetes, Pancreas, glis3, Insulin, Islets, Zebrafish

Thesis Advisor

Gary T ZeRuth

Committee Chair

Gary T ZeRuth

Committee Member

Alexey Arkov

Committee Member

David R Canning

Committee Member

J Ricky Cox

Committee Member

Chris Trzepacz

Document Type

Thesis

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