Multiple gut hormones are involved in the regulation of food intake. Secretin (SCT), a classical gut hormone, is released into the circulation from the duodenal S-cells when acidic chyme enters the duodenum and performs the major functions of delaying gastric emptying, stimulating fluid secretion from pancreas and liver to optimize the digestion process. In recent years, SCT and its receptor (SCTR) have been identified in discrete nuclei of the hypothalamus, including the paraventricular nucleus (PVN) and the arcuate nucleus (Arc). The occurrence of SCT and SCTR in the brain regions that are engaged in regulating body energy homeostasis and the release pattern of SCT after meals support a functional role of SCT in appetite control. In this study, the effect of SCT on feeding behavior was investigated using wild-type (wt), SCT?/?, and SCT receptor-deficient (SCTR?/?) mice. We found that both central and peripheral administration of SCT could reduce food intake in wt but not in SCTR?/?mice. SCT induce Fos expression in the PVN and Arc, suggesting the activation of hypothalamic feeding centers by this peptide. Consistent with this notion, SCT was found to increase proopiomelanocortin (POMC), but reduce agouti-related protein (AgRP) transcripts in the Arc, and augment thyrotropin-releasing hormone (TRH) and melanocortin-4 receptor (MC4R) mRNA expression in the PVN. In addition, pretreatment with SHU9119, an antagonist for MC4R, abolished the anorexia induced by SCT, suggesting that SCT may inhibit food intake via a melanocortin-dependent pathway. Gut hormones signals the brain to modulate the feeding behavior via the vagal afferent nerve, bloodstream or both. Here we showed that peripheral SCT-induced anorexia was attenuated in mice with subdiaphragmatic vagotomy, capsaicin treatment and bilateral midbrain transections. In summary, our data identify peripheral SCT as an anorectic peptide exerting its action via the melanocortin system and the vagal afferent contributes a major route in mediating the inhibitory effect of peripheral SCT on food intake. The present findings advance our understanding of the role of gut hormones in the regulation of appetite. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Secretin, the first hormone commencing the field of endocrinology, has been studied for its pleiotropic role in the body inclusive of its neuroactive and body water homeostatic and gastrointestinal functions. Yet, the metabolic effect of secretin remains elusive and is being proposed recently for a revisit. Recent discovery from our lab showed an anorectic response for secretin, while its role in lipid homeostasis remains largely unexplored. Exerting functions such as exocrine pancreatic secretion and gastric motility inhibition, intestinal fatty acid induced release of secretin was recently shown to be mediated by CD36. Fasting related increase in plasma secretin concentration has been proposed to be involved in lipolysis but evidences regarding lipolytic actions of secretin remain contradictory. Recent report has suggested that secretin stimulates both lipolysis and lipogenesis in adipose cells. Thus, we hypothesize that secretin modulates lipid homeostasis, which was examined under two opposite, energy deficient and energy excess, conditions. Under energy deficient/starved state, secretin level in circulation and secretin receptor level in epididymal adipose tissue were found to be upregulated. Using secretin receptor knockout (SCTR-/-) and secretin knockout (SCT-/-) mice as controls, it was found that secretin stimulated a dose- and time-dependent lipolysis in vitro and acute lipolysis in vivo. H-89, a protein kinase A (PKA) inhibitor, attenuated the lipolytic effects of secretin in vitro, while secretin induced an increase in cAMP dependent PKA activity in vivo. Using western blot analysis, secretin was found to phosphorylate hormone sensitive lipase (HSL) at serine residue 660. Additionally, immunofluorescent studies revealed that secretin stimulated translocation of HSL from cytosol to surface of lipid droplet subsequently leading to lipolysis. Under excess energy condition, when SCTR-/- mice and its littermates SCTR+/+ mice were subjected to high fat diet (HFD) feeding for 3 months, it was found that SCTR-/- mice gained lesser weight. Nuclear magnetic resonance imaging revealed that SCTR-/- mice exhibited lower body fat content. Additionally, HFD-associated hyperleptinaemia was alleviated in SCTR-/- mice along with metabolic syndrome as they performed better in insulin and glucose tolerance tests. Continuous monitoring by indirect calorimetry revealed similar food intake, energy expenditure and locomotor activity between SCTR-/- and SCTR+/+ mice. Interestingly, intestinal fatty acid absorption, measured by a noninvasive method, was impaired in HFD-fed SCTR-/- mice. While postprandial triglyceride release was reduced in SCTR-/- mice, it also had a significant reduction in transcript and protein levels of CD36 and its downstream mediator MTTP. Secretin, when incubated with isolated enterocytes, upregulated the expression of CD36. In summary, during starvation, secretin stimulates lipolysis through a HSL and PKA mediated pathway. When fed a HFD, SCTR-/- mice is resistant to diet induced obesity due to impaired intestinal lipid absorption. A novel short positive feedback pathway between CD36 and secretin, functioning to maximize lipid absorption, is also being proposed. Thus for the first time, two independent role of secretin in lipolysis and in intestinal lipid absorption were discovered along with their mechanistic insights. This study paves way for developing new therapeutic strategies against metabolic disorders associated with lipid metabolism. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Class B GPCRs are emerging target in drug research. Currently these receptors serve as drug targets for several drug discovery companies and more than 50 percent of the drugs in the market targets GPCRs. Secretin receptor is found to be expressed in various tissues. Secretin regulates many bodily functions from energy to water homeostasis through both central and peripheral system. Though it holds a history of 100 years, the major drawback is its structural insights. In evidence of its integrated role in physiology as a potential target, the lookout for a novel agonist and / or antagonist for secretin receptor is initiated. As this target is in the primary state of drug research, it is also necessary to develop the appropriate screening platforms. Due to the lack of experimental structure of secretin receptor-ligand, a 3D virtual homology model is developed using multiple template approach. Besides virtual docking, a non-radioactive FRET competitive binding assay is also developed and substantiated to enable the receptor-ligand interaction studies. Both peptide and non-peptide analogs were screened for virtual docking, in vitro binding and functional response. For the peptide analogs, the modifications were made either in the N or C terminal portion of the peptide based on the previous findings that C-terminal portion is involved in receptor binding followed by allosteric modifications and N-terminal portion is involved in activation. These peptide analogs exhibited binding affinity in the virtual model. Paradoxically it did not exhibit in vitro binding as predicted. Along with this, the agonistic and antagonistic functional responses of these peptide analogs were also found to be negative. SPECS natural product library of 500 non-peptide analogs were screened virtually against secretin receptor and 32 hits were identified. Of these hits glycyrrhizin’s functions were comparable to secretin was screened for receptor binding and functional response. These in vitro assays did not exert anything positive; however an IP-GTT on WT, 〖SCT〗^(-/- )and 〖SCTR〗^(-/-) mice with acute treatment of glycyrrhizin at 10 mg/kg and chronic treatment of 5 mg/kg exhibited an interesting profile with negligible effect on 〖SCT〗^(-/- )mice whereas in WT and 〖SCTR〗^(-/-) mice it displayed a better profile with improved glucose tolerance. The chronic study serum analysis on day 28 exhibited substantial reduction in blood glucose while significant increase in serum secretin and insulin levels. As glycyrrhizin promotes secretin secretion, its acute effect on blood pressure in WT mice was also analyzed at 10 mg/kg; remarkably exhibited a significant drop in blood pressure. In summary modifications in the peptide analogs lead to instability in the receptor-ligand binding complex in the in vitro system leading to loss of binding efficiency. In case of non-peptides, though glycyrrhizin could not exhibit in vitro response, its supplementary mechanism through secretin pathway of increased secretin release is confirmed using the WT, 〖SCT〗^(-/- )and 〖SCTR〗^(-/-) mice. The hypotensive effect with an acute treatment in WT is also revealed. Discovery of this new mechanism of an old drug could broaden the research for a new class of drug, “secretin sensitizers / promoters”. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Motor coordination and motor learning processes are vital for animal survival. Both functions require the participation of cerebellar Purkinje neurons, which are the integrating center as they receive both excitatory and inhibitory inputs from various neurons and send out the sole inhibitory output of cerebellar cortex. Secretin, a classical peptide hormone, has been shown previously as a retrograde factor to up-regulate GABAergic inhibitory transmission on basket-Purkinje cell synapses in rats. In behavioral perspectives, there have been studies reporting changes of motor function, anxiety level, spatial memory and social interactions after application or deprivation of secretin. Based on current knowledge, secretin is hypothesized to modulate mouse motor coordination and motor learning behaviors through its effects on Purkinje neurons. To test this hypothesis, a Purkinje cell-specific secretin gene knockout moue model (Pur-Sct-/-) has been developed using Cre-Loxp recombination technique. Using immunohistochemical staining and in situ hybridization, secretin expression has been shown to be specifically eliminated in Purkinje neurons. Pur-Sct-/- mice had intact general motor ability and anxiety level in an open field. Neuromuscular strength of Pur-Sct-/-mice was impaired compared to wild type littermates in wire hanging test. Motor coordination ability was compromised as shown by vertical climbing and rotarod tasks. Further tests using repeated training on rotarod suggested impaired motor learning ability. All these behavioral changes have also been observed in secretin-null (Sct-/-) and secretin receptor-null (Sctr-/-) mice, suggesting that normal motor control and motor learning depend on integrity of secretin-secretin receptor axis in cerebellum. Postnatal neural developmental study revealed later occurrence of two motor reflexes –righting and negative geotaxis –in Pur-Sct-/-juveniles. Motor deficits in rotarod tasks persisted across mice aging from month 2 to month 9 while wire hanging impairments occurred early in Pur-Sct-/-. Secretin thus may also play a role in early postnatal cerebellar development and neural protection in mature cerebellum. To investigate the underlying mechanism, Purkinje neurons were voltage-clamped for current recording. Secretin potentiated both spontaneous and miniature inhibitory postsynaptic currents (sIPSC and mIPSC) in wild type Purkinje cells. InPur-Sct-/- and Sctr-/- mice, basal levels of sIPSC and mIPSC were significantly decreased, suggesting a role of endogenous secretin in maintaining cerebellar inhibitory transmission. The exogenous application of secretin restored IPSC in Pur-Sct-/- but not in Sctr-/- mice to comparable wild type levels, indicating the specific binding of Purkinje-derived secretin and secretin receptor underlyingthis inhibitory potentiation. In summary, secretin released in Purkinje neurons has significant role in maintaining normal motor coordination and motor learning functions. Secretin also participates in the facilitation of inhibitory transmission on interneuron-Purkinje synapses. This inhibitory potentiation is likely to coordinate motor behaviors, although further in vivo studies are required for substantiation. This study has demonstrated the function of secretin in modulating mouse motor coordination and motor learning behaviors, and in Purkinje neuron inhibitory transmission, suggesting its potential usage in drug development against cerebellar-related motor disorders. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 117-150) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 117-150) Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 55-68).

Thesis (M.Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 55-68) Also available in print.