Whether vascular endothelial growth factor A (VEGF) plays an augmented role during pregnancy is unknown. In this thesis expression of VEGF in the placenta, maternal circulation and organs in mice was examined using qRT-PCR, LacZ expression, and/or ELISAs. Normal pregnancies and pregnancies with transgenic conceptuses that over-express VEGF in the placenta were examined. In normal pregnancies, VEGF120/164 levels in the ovary increased in parallel with that of the maternal circulation. In pregnancies where the placenta over-expressed VEGF, maternal circulating VEGF120/164 levels decreased and so did levels in the maternal ovary. Surprisingly, VEGF protein levels (per mg of total protein) decreased in the growing, highly vascular placenta during pregnancy. In conclusion this thesis provides evidence for an important ovarian source of maternal circulating VEGF120/164 during pregnancy.

The hippocampus is a brain region that is critically involved in memory formation. Stratum oriens-lacunosum/moleculare (O-LM) interneurons have been shown to modulate incoming sensory information onto principal cells in CA1. Multi-compartment computational models of O-LM cells have been developed to better understand their functional roles in network contexts. Due to the variability and incompleteness of experimental details, however, a population of models that collectively captures intrinsic O-LM cell behavior is needed. We generated a database of O-LM models with physiologically plausible ranges for conductance densities using NEURON simulations on a supercomputer cluster. A subset of models that best represented O-LM cell electrophysiological output was subsequently extracted from the database and analyzed in order to determine correlations in conductance densities. Three major co-regulatory balances were found, which provide specific hypotheses for experimental investigations and point to the possibility of identifying a “signature” of conductance density balances for particular neuronal cell types.

Neuronal vulnerability to ischemia is dependent on the balance between pro-survival and pro-death cellular signaling. In the latter, it is increasingly appreciated that toxic Ca2+ influx can occur not only via postsynaptic glutamate receptors, but also through other cation conductances. One such conductance, the Transient receptor potential melastatin type-2 (TRPM2) channel, is a non-specific cation channel having similar homology to TRPM7, a conductance reported to play a key role in anoxic neuronal death. The role of TRPM2 conductances in ischemic Ca2+ influx has been difficult to study due to the lack of specific modulators. Here we used TRPM2-null mice (TRPM2(-/-)) to study how TRPM2 may modulate neuronal vulnerability to ischemia. TRPM2(-/-) mice subjected to transient middle cerebral artery occlusion (tMCAO) exhibited smaller infarcts when compared to wild-type (WT) animals, suggesting the absence of TRPM2 to be protective. Surprisingly, field potentials (fEPSPs) recorded during oxidative stress in brain slices taken from TRPM2(-/-) mice revealed increased excitability, a phenomenon normally associated with ischemic vulnerability, whereas WT fEPSPs were unaffected. The upregulation in fEPSP in TRPM2(-/-) neurons was blocked selectively by an NR2A antagonist. This oxidative stress-induced increase in excitability of TRPM2(-/-) fEPSPs depended on the upregulation and downregulation of NR2A and NR2B-containing NMDARs, respectively, and augmented pro-survival signaling via Akt and ERK pathways culminating in the inhibition of the proapoptotic factor, GSK3β. Cultured hippocampal neurons from TRPM2(-/-) animals subjected to oxygen glucose deprivation had a reduction in cell death in comparison to WT neurons, demonstrating that absence of TRPM2 is protective at the neuronal level in vitro. Our results suggest that TRPM2 plays a role in downregulating pro-survival signals in central neurons and that TRPM2 channels may comprise a therapeutic target for preventing ischemic damage.

The two of major pathways that cause ischemic damage are oxidative stress and inflammation. To decreasing oxidative stress and inflammation, new anti-oxidant and anti-inflammatory agents are tested in ischemic models. In order to study ALRX828C anti-inflammatory properties, an in vivo six-day old air pouch model of inflammation was used to evaluate the anti-inflammatory potential of ALRX828C. Also, the dose response of ALRX828C for TNFα (IC50 = 30 μM) and IL-17 (IC50 = 1.3 μM) were determined by using human peripheral blood mononuclear cell cultures stimulated with ionomycin and PMA. To examine ALRX828C anti-inflammatory effect in neuroinflammation, a neurodegenerative model was used to evaluate its potential. I also showed that reducing oxidative stress with a potent antioxidant, Idebenone in nano-emulsion form, can effectively reduce tissue damage during ischemia in organotypic slice culture subjected to oxygen-glucose depravation (OGD). In conclusion, reducing oxidative stress and inflammation after stroke can reduce ischemic damage substantially.

Regulators of G-protein signaling (RGS) modulate G-protein coupled receptor (GPCR) activity in vascular smooth muscle cells (VSMCs). One such protein, RGS5, has been shown to have selective expression in VSMCs and pericytes, and can inhibit signaling from Gαq and Gαi subunits. Using an RGS5 knockout model, we assessed the functional effect of RGS5 in the constriction and dilation of resistance arterioles. Furthermore, we examined the intracellular lipid interaction of RGS proteins to identify the determinants regulating the biologic function of RGS5. Surprisingly, loss of RGS5 function in mesenteric arterioles had no effect on constriction and dilation of resistance arterioles. Cultured VSMCs showed increased basal ERK1/2 phosphorylation and increased VASP signaling in response to SNP treatment in RGS5KO VSMCs as compared to wild type controls, with no effect on cell proliferation. These data suggest RGS5 may integrate multiple intracellular pathways with competing effects on VSMC contraction.

Nutrient sensing pathways in both the brain and gut decrease hepatic glucose production. Hypothalamic activation of lactate metabolism decreases glucose production, but it is unknown whether the hypothalamus detects circulating lactate to maintain glucose homeostasis. In the gut, lipids decrease glucose production via a neuronal network but the downstream signaling mechanisms are unknown. We tested whether circulating lactate activates central lactate metabolism to decrease glucose production and postulated that duodenal protein kinase C (PKC) acts downstream of lipids to decrease glucose production through a neuronal network. We report that central lactate metabolism is required for the maintenance of glucose homeostasis in the presence of circulating lactate and that activation of duodenal PKC is required for lipids to decrease glucose production. This shows the importance of the brain and gut in the regulation of glucose production, and could pave the way for restoration of glucose homeostasis in disease.

Cholecystokinin (CCK) is a gut peptide involved in the regulation of energy homeostasis by duodenal lipids via a neuronal network. However, it is unknown whether CCK also regulates glucose homeostasis through a neuronal network. Using an in vivo rat model, we demonstrated that duodenal CCK-8 (biologically active form of CCK) can lower glucose production through the activation of a gut-brain-liver axis via CCK-A receptors, and this glucose-regulatory effect is physiologically relevant. Since duodenal lipids can also lower glucose production through a gut-brain-liver axis, we verified that this duodenal-lipid effect is mediated by CCK-A receptor activation. Lastly, in rats fed on a high-fat diet for three days, duodenal CCK failed to suppress glucose production, suggesting a state of CCK-resistance. In summary, these findings revealed that intestinal CCK can regulate glucose homeostasis through a neuronal network and suggest that intestinal CCK resistance may contribute to hyperglycemia in response to high-fat feeding.

The CAPS1 protein was initially discovered as a cytosolic soluble 145kDa protein which was necessary to restore calcium dependent norephinephrine secretion in cracked PC12 cells. Recent findings suggest that CAPS may also play a role in synaptic and dense core vesicle exocytosis as well as in the loading of monoamine neurotransmitters. Recently, studies have implicated CAPS1 in the binding to syntaxin-1. However, no studies have identified the key residues of CAPS1 that facilitate this interaction with syntaxin-1. I show that the binding mode of CAPS1 is independent from that of Munc13 such that CAPS1 requires the full length of syntaxin-1 to bind. Moreover, CAPS1 favors the open conformation of syntaxin-1. Interestingly, the Munc homology (MH) domain of CAPS1 is not critical for this interaction while the C-terminal dense core vesicle binding (DCVB) domain plays an important role. Moreover, truncations to this DCVB domain result in decreased binding to syntaxin-1.

The physiological mechanisms that control energy homeostasis are reciprocally linked to reproduction. However, the neuroendocrine circuitry that registers endocrine cues to direct homeostatic responses in energy balance and reproduction remain unknown. Neuropeptide Y (NPY) neurons have emerged as a key central target of estrogen and leptin that are capable of modulating both reproduction and energy balance. The hypothesis was generated that NPY neuronal subpopulations act as an integration centre to regulate the effects of estrogen and leptin on these important physiological processes through specific signaling pathways. Using hypothalamic cell lines that express the leptin receptor (Ob-R), estrogen receptor (ER) and NPY, this hypothesis was tested in three aims. 17β-estradiol (E2) was previously demonstrated to biphasically regulate NPY mRNA in the mHypoE-38 neuronal cell line; where 24 h E2 exposure induced NPY gene expression that our group proposed may be involved in the gonadotropin-releasing hormone (GnRH) preovulatory surge. E2 also acts as an anorexigenic hormone through unknown hypothalamic targets. E2 directly decreased NPY secretion in the mHypoE-42 and mHypoA-2/12 neuronal cell lines through ER-α. The anorexigenic action of E2 was mediated through the energy sensing 5’ AMP-activated protein kinase (AMPK) and the phosphoinositide-3-kinase (PI3K) pathway. NPY secretion was also decreased by leptin in mHypoA-59 and NPY-GFP cell models through AMPK- and PI3K-dependent mechanisms. Prolonged exposure to leptin in NPY-GFP cell lines prevented AMPK signaling and the leptin-mediated reduction in NPY secretion, indicating NPY neuronal resistance with prolonged leptin exposure. Leptin also stimulated NPY secretion in mHypoE-38 neurons, which was blocked by pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) and PI3K pathways. Importantly, conditioned medium from the mHypoE-38 NPY neuronal cells induced GnRH transcripts in GT1-7 neurons, which was inhibited by Y1-receptor antagonists. Pharmacological inhibitors of the MAPK and PKA signal transduction pathways attenuated the NPY-mediated increase in GnRH transcription. Based upon these findings, I propose NPY neurons in the hypothalamus consist of a heterogeneous population of neurons, and provide the first evidence of intrinsically different responses to function as physiological integrators for two different systems: NPY secretion can be suppressed to decrease food intake and induced to stimulate GnRH neurons.

GLP-2 is a potent intestinotrophic peptide that can increase mucosal growth, intestinal blood flow, and nutrient absorption when administered exogenously. We aimed to delineate the effects of endogenous GLP-2R signalling in conditions of nutrient deprivation and excess. Using a mouse with a targeted genetic deletion of the Glp2r gene (Glp2r-/-), we addressed the hypothesis that the known GLP-2R is required for intestinal adaptation to nutrient deprivation and excess. In Chapter 2, we demonstrate that Glp2r−/− mice fasted for 24 hours and re-fed for 24 hours failed to increase intestinal growth and jejunal crypt cell proliferation compared to littermate Glp2r+/+ mice. Administration of EGF to Glp2r−/− during the re-feeding period rescued this re-feeding defect. Wildtype mice re-fed for 30, 90, and 180 minutes following a 24 hour fast displayed increased jejunal mRNA levels of the ErbB ligands amphiregulin, epiregulin and HB-EGF. Treatment with the pan ErbB inhibitor CI-1033 inhibited induction of these ErbB ligands in jejunum of mice in association with prevention of crypt cell proliferation. Re-feeding also caused an increase in jejunal p-Akt levels and treatment with CI-1033 prevented increased p-Akt levels. Moreover, re-fed Glp2r−/− mice failed to increase ErbB ligands or p-Akt levels 90 minutes following re-feeding when compared to Glp2r+/+ littermates. Therefore, the GLP-2R is essential for re-feeding induced intestinal adaptation by activating the ErbB network and p-Akt to increase crypt cell proliferation. In Chapter 3, we show that the known GLP-2R is not required for intestinal adaptation to a perceived nutrient deprivation challenge (STZ-induced diabetes) or chronic nutrient excess (high-fat diet induced glucose intolerance). Although exogenous GLP-2 administration has been previously shown to stimulate glucagon secretion, glucose homeostasis was normal in STZ-diabetic and high fat fed Glp2r−/− mice. We also developed a third model of diabetes and glucose intolerance: ob/ob: Glp2r−/−. In the absence of GLP-2R signalling, ob/ob mice display improved oral but impaired intraperitoneal glucose tolerance, elevated fed and fasted glucose levels, increased circulating glucagon, decreased beta cell and increased alpha cell mass. Taken together, these results suggest that endogenous GLP-2R signalling is essential for intestinal and islet adaptation to conditions of nutrient deprivation and excess.