Hypothalamic AMP-activated protein kinase (AMPK) regulates energy homeostasis in response to nutritional and hormonal signals. However, its role in glucose production regulation remains to be elucidated. Here, we tested the hypothesis that bidirectional changes in hypothalamic AMPK activity alter glucose production in rodents. First, we found that knocking down hypothalamic AMPK activity in an in vivo rat model led to a significant suppression of glucose production independent of changes in food intake and body weight. Second, we showed that activation of hypothalamic AMPK negated the ability of hypothalamic glucose- and lactate- sensing to lower glucose production. Collectively, these data indicate that changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to alter glucose production in vivo, and highlight the novel role of hypothalamic AMPK in the maintenance of glucose homeostasis in addition to energy balance.

The incidence of preterm labor has risen over recent decades and preventative antibiotic treatment is ineffective. Associated with a 40% increased risk of preterm birth, bacterial vaginosis is characterized by a decrease in lactobacilli and increase in pathogenic bacteria in the vaginal flora. Ascent of bacterial products to the intrauterine environment stimulates cytokine and prostaglandin secretion from invading immune cells and gestational tissues. Probiotic lactobacilli modulate the immune responses in mouse macrophages and human placental trophoblast cells. The focus of this thesis was to determine the influence of Lactobacillus rhamnosus GR-1 (GR-1) on cytokines and prostaglandins which are part of the activated pathway in infection and/or inflammation mediated preterm labour. GR-1 increased amnion chemokine and reduced pro-inflammatory cytokine release. GR-1 elevated prostaglandin E2 release that was paralleled by an increase in mPGES2 expression. It is possible that t that GR-1 may enhance the host defense barriers of the amnion to pathogenic bacteria.

Renal cell carcinoma (RCC) is the commonest urogenital tumor, characterized by increased expression of hypoxia inducible factors (HIFs). During normoxia, HIFα subunits are targeted for proteasomal degradation by the product of the von Hippel Lindau gene (pVHL). In RCC, mutations in the VHL gene allow the HIFα subunits to escape degradation and translocate to the nucleus where they activate transcription of their target genes. Although both HIF1α and HIF2α are upregulated in RCC, it has been suggested that HIF2α plays the dominant role. To further elucidate the function of HIF2α in RCC, we generated a transgenic mouse model that permits temporal stabilization of HIF2α in renal tubular cells. Induction of HIF2α results in the rapid development of renal cysts - a feature observed in RCC. Taken together, these results suggest that HIF2α is a key player in development of RCC and an excellent candidate target for therapy in this disorder.

Mice (C57BL/6J: B6) exposed to maternal dietary restriction (DR) exhibited fetal growth- restriction and as adults develop symptoms of the metabolic syndrome. We aimed to determine the impact of DR on fetal hepatic gluconeogenic pathway and insulin sensitivity in late gestation. Second, we aimed to determine whether a postweaning diet rich in omega-3 fatty acids would alter the development of glucose intolerance, insulin resistance and obesity in DR male offspring. The reduced rate of fetal glycogen synthesis by DR male offspring and altered hepatic gene expression of enzymes involved in insulin signalling and glucose metabolism suggest abnormal fetal development in response to DR that may contribute to the later development of the metabolic syndrome. The postweaning omega-3 diet improved obesity, glucose intolerance and insulin resistance in both DR and control males. These data suggest that nutrition in pregnancy and postnatal life play important roles in determining life-long metabolic health.

The cWnt activator, Rspondin-1 (Rspo1), has been identified as a regulator of β-cell growth and function, although its role in pathophysiological conditions such as streptozotocin (STZ)-induced diabetes is unknown. Hence, I hypothesized that Rspo1 deficiency stimulates β-cell neogenesis in STZ-diabetes. There was no difference in oral glucose handling between STZ-induced Rspo1mice, although, Rspo1-/- mice demonstrated increased insulin sensitivity compared to wild-type littermates. Moreover, β-cell mass and the total number of islets did not differ between STZ-induced Rspo1+/+ and Rspo1-/- mice, although mice with Rspo1 deficiency had reduced β-cell apoptosis and significantly enhanced numbers of insulin-positive ductal cells suggestive of β-cell neogenesis. Furthermore, the increased β-cell regeneration observed in knockout animals appeared to be associated with a more differentiated/mature β-cell phenotype in Rspo1-/- versus Rspo1+/+ mice. Collectively, these findings indicate a role for Rspo1 as a negative regulator of in vivo β-cell neogenesis and survival in the face of STZ-induced diabetes.

Impaired counterregulation during hypoglycemia in type 1 diabetes (T1D) is partly due to inadequate pancreatic islet alpha-cell glucagon secretion. We hypothesized that hypoglycemia can be prevented in autoimmune T1D by selective somatostatin receptor type 2 (SSTR2) antagonism of alpha cells to relieve SSTR2 inhibition, thereby increasing glucagon secretion. Diabetic biobreeding diabetes prone (BBDP) rats (D) vs non-diabetic BBDP (N) rats, underwent infusion of vehicle or SSTR2 antagonist (SSTR2a) during insulin-induced hypoglycaemia. D rats, treated with SSTR2a, needed little or no glucose to maintain hypoglycemia. To monitor real-time glucagon secretory response directly, we developed the technique of thin slices of the pancreas from D and N rats as well as normal human pancreas, subjected to perifusion with vehicle vs SSTR2a. SSTR2a treatment enhanced glucagon secretion in N and D rats and human pancreas. We conclude that SSTR2 antagonism can enhance hypoglycemia-stimulated glucagon release sufficient to achieve normoglycemic control.

Discovering novel compounds that stimulate or abrogate angiogenesis can lead to development of new therapeutic agents that may effectively treat diseases with pathological angiogenesis. The zebrafish model allows for a whole-organism approach to drug discovery. Advantages over other animal models include small embryo size, fecundity, rapid embryonic development, optical clarity and easy accessibility of the embryos. My goal is to validate the therapeutic efficacy and identify the molecular mechanisms of action of three compounds identified from our previous chemical genetic screens. Fenretinide promoted angiogenesis in zebrafish embryos but inhibited the angiogenesis-dependent process of fin regeneration. The pro-angiogenic effects of fenretinide appear secondary to the stimulation of somitogenesis. I3M potently inhibited angiogenesis and fin regeneration, and may act partially through the notch pathway. Lastly, I validated the anti-angiogenic effect of a novel compound DHM. Comprehensively, my studies support the utility of zebrafish as a versatile tool for anti-angiogenic drug discovery.

Microglia, the resident immune cells of the central nervous system, can become activated following injury, disease, or infection. In vitro, they can be activated by stimuli, which determine the inflammatory phenotype they will develop. In this thesis, stimulating microglia with tumor necrosis factor- and interferon- resulted in classical activation, characterized by proliferation, increased transcription of complement receptor 3 and major histocompatibility class II molecules, and elevated production and transcription of interleukin-1 and nitric oxide. Stimulation with TNF and IFN also changed the intensity of phosphorylated (activated) cyclic adenosine monophosphate response element binding protein immunoreactivity in microglia. Specifically, cells differentiated into populations with high or low pCREB intensity. This was the first example of such a response in microglia and was representative of what occurred in vivo, after ICH. Thus, the characterization of this model will be useful for future studies of this and other intracellular pathways of classically activated microglia.

Hedgehog (HH) signaling controls renal development. Mutations in PTC1, the HH receptor, cause cancer in non-renal tissues. We hypothesized that constitutively active HH signaling is deleterious to renal development in mice with PTC1 deficiency targeted to the metanephric mesenchyme (MM)(Rarb2-Cre;Ptc1 loxP/-, termed Ptc1 mutants). Increased HH signaling in MM of mutant mice was confirmed by qRT-PCR for Ptc1. A decrease in NCAM-positive nephrogenic precursors at E13.5 and WT1-positive glomeruli at E18.5 was found. Increased cortical expression of Foxd1 was observed. At E13.5, a cluster of ectopic cells expressing Raldh2, Ptc2 and Bmp4 accumulated at the presumptive uretero-pelvic junction (UPJ). Magnetic resonance imaging demonstrated an increase in pelvic volume. Constitutive expression of GLI3 repressor via the Gli3Δ699 allele in Ptc1 mutants increased nephron number comparable to wild type mice and decreased pelvic volume compared to Ptc1 mutants. Thus repression of HH activity is required for proper nephrogenesis and patterning of the UPJ.

Bladder obstruction (neurogenic or anatomic) induces strain injury in detrusor smooth muscle cells. Signaling via strain injury in other systems has been highly studied, while in bladder obstruction, it has been quite limited to a small number of pathways. In our study we have examined the effects of strain injury using a combination of in vivo, ex vivo and in vitro models, with the aim of understanding disease pathogenesis in the bladder. Using a combination of literature searches, phospho-protein screens and pathway analysis, we uncovered three pathways activated by mechanical strain, ERK, STAT3 and mTOR, with potential for changing not only the way we understand but also the way we treat obstructive myopathies of the bladder. We found that not only were these pathways activated in response to strain and distension injury of BSMC, but they were also responsible for proliferation and sometimes de-differentiation. Included herein are three chapters, published in 2006 and 2010, on the role of ERK, STAT3 and mTOR pathways in bladder smooth muscle cell proliferation and differentiation, 8 Appendices containing the first pages of other papers and reviews published during the course of my studies.