|dc.description.abstract||Obesity and type 2 diabetes mellitus (T2DM) are among the most common and costly health-related issues worldwide, and both have been declared a global epidemic by the World Health Organization. A key characteristic of these conditions is disturbed leptin signalling. Leptin, named after the Greek word for ‘lean’ (λεπτός), is a hormone that plays a key role in energy homeostasis. Although known best for its adipostatic role in mammals, leptin also regulates glucose homeostasis independent of effects on adipostasis. To date, the mechanism through which leptin exerts its glucoregulatory actions remains largely unknown. This thesis investigates whether leptin regulates glucose homeostasis via the canonical WNT pathway in the zebrafish (Danio rerio). Non-mammalian leptin studies have been conducted for only a little bit over a decade, and still comprise but a tiny fraction (<2%) of the total research in this field. However, these studies are needed not only to answer comparative questions, but uncovering the evolutionary origins of leptin function will clarify our understanding of how this hormone functions in the human body.
Using the CRISPR/Cas9 mutagenesis system, stable mutant zebrafish lines were successfully generated (chapter 2). These fish had a homozygous knockout of either the leptin-a gene (lepa), the leptin-b gene (lepb) or the leptin receptor gene (lepr). Next, protocols were established to investigate glucose homeostasis in adult zebrafish (chapter 3). Existing procedures for intraperitoneal injection and blood sampling were adapted to create a protocol for intraperitoneal glucose tolerance tests in adult zebrafish. In addition to this acute hyperglycaemic challenge, a glucose-immersion protocol was established to allow for the investigation of artificially-induced persistent hyperglycaemia as well.
Combining the mutant fish generated in chapter 2 with the protocols established in chapter 3, the effects of leptin- and leptin receptor deficiency were characterized in adult zebrafish (chapter 4). It was confirmed that under normal feeding conditions, leptin regulates glucose homeostasis but not adipostasis in the zebrafish. However, in times of nutrient excess leptin was found to regulate body weight and standard length, and glucose homeostasis was impaired.
Next, the canonical WNT pathway was investigated as a potential mediator of the glucoregulatory effects of leptin. Taking advantage of the optical transparency of zebrafish larvae, a transgenic zebrafish line expressing fluorescence upon WNT pathway activation was used to demonstrate that leptin directly activates the canonical WNT pathway in vivo, specifically in the hypothalamus (chapter 5). Finally, various pharmacological manipulations of the canonical WNT pathway were performed in leptin-mutant fish and wild type controls to demonstrate that leptin regulates glucose homeostasis via the canonical WNT pathway (chapter 6).
Together, these findings show a novel essential role of the canonical Wnt pathway in the neuroendocrine control of glucose homeostasis in zebrafish. Furthermore, these data highlight that leptin may primarily have evolved as a glucoregulatory hormone with its role of an adipostat acquired later in evolution. Finally, the glucoregulatory action of leptin is mediated via the Wnt pathway - an essential mechanism that appears preserved throughout the vertebrate phylum.||