The present thesis examined the potential interaction between postingestive cues and the dopamine antagonist pimozide on the inhibition of sucrose sham intake. Coadministration of CCK and pimozide IP produced additive inhibitory effects on sham feeding of 4% or 17.1% sucrose. Manipulations of blood glucose levels by infusion of 10% dextrose or injection of 0.1U insulin SC did not interact with pimozide to alter the latter drug's inhibition of 34.2% sucrose sham feeding. Separate experiments verified that these manipulations significantly altered blood glucose levels compared to a control 0.9% saline infusion IV. Infusions of d-glucose into the lateral ventricle significantly enhanced the inhibitory effects of pimozide on 34.2% sucrose sham intake compared to infusions of!glucose or 2-deoxy-glucose. Infusions of d-glucose into the third ventricle, however, significantly attenuated the inhibitory effects of pimozide on 34.2% sucrose sham feeding compared to the control l-glucose infusions. These results collectively suggest that some postingestive cues, such as elevations in third ventricle glucose levels, are indeed capable of inhibiting the suppressive effects of pimozide on sucrose sham feeding. / Thesis / Master of Science (MSc)

Thesis advisor: Barbara E. Wolfe / The core defining features of bulimia nervosa (BN) are repeated binge eating episodes and compensatory purging behavior. The biobehavioral aspects of binge eating are complex and not fully understood. One area of recent interest is the role of the satiety-signaling peptide cholecystokinin (CCK). Previous research observed a blunted postprandial plasma CCK response in those with BN, therefore suggesting this may be a cause, consequence, or maintenance factor in binge eating. It is unknown whether this altered response is due to a state versus trait phenomenon, thus having implications in the development of clinical treatment strategies. To examine the nature of this altered response, this study assessed whether CCK normalizes following remission from BN (RBN). This biobehavioral study utilized a comparative design to prospectively evaluate the biological CCK response and the corresponding behavioral ratings of satiety and other eating-related sensations in individuals with BN (n=10), RBN (n =14), and healthy controls (CON, n=13). CCK and behavioral ratings were assessed at baseline, +15, +30, and +60 minutes following the ingestion of a standardized liquid test meal. The BN group's CCK response was blunted and approached significance (p =.052) when compared to the RBN and CON groups. As predicted the RBN and CON groups' CCK response did not significantly differ. This finding supports the premise that CCK may normalize following abstinence from binge and purge (vomit) episodes and that this is a state versus trait related phenomenon. A significant positive relationship between CCK response and ratings of satiety occurred in the RBN group only (r=.59, p<.05). A new and unanticipated finding in the BN group was a significant relationship (r=.86, p < .01, two-tailed) between their CCK response and urge to vomit. A greater urge to vomit was reported by those individuals who had increased CCK response. Therefore, it is unknown whether the normalization of CCK functioning is a protective or liability factor in the stabilization and recovery process. Replication studies utilizing a larger sample size are needed to understand the role of CCK in recovery and the subsequent development of novel treatment strategies for those suffering with BN. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Connell School of Nursing. / Discipline: Nursing.

The central nervous system (CNS) has a crucial role in the maintenance of energy homeostasis by orchestrating a plethora of signals from peripheral organs about the state of energy stores and the current energy intake needed to match energy expenditure. These signals converge into the hypothalamic regions and its complex local circuitry. CNS-derived cholecystokinin (CCK) is acting at central level to modulate energy balance by regulating the neuronal activity of hypothalamic neuronal populations that regulate food intake, energy storage and consumption. Moreover, our recent published work identifies CCK neurons as key integrators of the neuroendocrine negative feedback of glucocorticoids to the PVN. Glucose sensing neurons of the Ventromedial Hypothalamus (VMH) are integrating energy signals and are essential for mounting a counter-regulatory response and glucose homeostasis. VMH is also important in energy expenditure by regulating body weight and thermogenesis. CCK neurons are present in high density in the VMH.The source of endogenous CCK that acts on distinct neuronal components has not been elucidated. The research so far does not address the purpose of CCK neurons in the hypothalamus and their potential role in the network dynamics regarding energy homeostasis. In this study, we untangle the role of CCK neurons in the VMH nucleus by employing stereotactic intracranial delivery of adeno-associated viruses that result in cell-type specific chemogenetic inhibition or ablation of these neurons. Acute silencing of their neurotransmission with the cre-dependent AAV expression of the chemogenetic tool of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) increases their daily food intake due to increased meal numbers and eating frequency without meal size or meal duration being affected. CCK ablation by a newly generated double-recombinase-mediated Diphtheria Toxin Receptor (DTR) mouse line or AAV-DTA-mediated ablation resulted in hyperphagia, obesity and hyperglycaemia. We conclude that CCK^VMH neurons are implicated in the regulation of food intake, body weight and glucose homeostasis in the adult brain.

Cholecystokinin (CCK) secreting enteroendocrine (EEC) I cells which distribute in gastrointestinal tract play an important role in lipid sensing, digestion and fatty acids uptake. Although a lot of research has been performed, the whole mechanism of fat sensing and fatty acid uptake and hormone expression in the CCK cells is still unclear. Global analysis to characterise the CCK cells is essential. CCK cells have an indistinct morphology, a diffuse distribution and a small percentage of population in the small intestine. However, the generation of genetic fluorescence tagged animal model facilitates the study of these cells. In this thesis, single cell dissociation methods and RT-PCR methodologies for detecting nutrient sensing receptors and fatty acids transporters were established and optimised. Expression of mRNAs for fat sensing GPCRs were detected in mouse duodenal epithelium. Expression of FATP family and CD36 in CCK cells and enterocytes was studied by RT-PCR. FATP2, FATP4 and CD36 mRNA were found in both CCK cells and enterocytes. Cell culture methodologies enabling the study of function (calcium imaging and FACS analysis) were established and optimised by checking the cell viability as a criterion. The methodology combining the immunochemistry and FACS analysis to study the hormone was established but requires further optimisation.

The research presented in this thesis relates to the gastrointestinal control of appetite and some of the consequences of nutritional frailty, namely postprandial hypotension and vitamin D insufficiency. Undernutrition and its consequences are increasingly common problems in an ageing population, and improved management is dependent on an understanding of the factors which are involved in the control of appetite, and the physiological decline of appetite with increasing age termed ‘the anorexia of ageing’. The role of the gastrointestinal hormone ghrelin was specifically evaluated, in relation to the effects of age and nutrient digestion on circulating ghrelin concentrations (Chapters 6 and 7). The effect of fat digestion on the postprandial blood pressure response in healthy older subjects was evaluated in the study reported in Chapter 8. In addition, the results of some intervention studies are described in Chapters 9 and 10, the former study relating to nutritional supplementation as a strategy to increase energy intake, and the latter study to the effects of vitamin D replacement therapy on glucose and insulin metabolism. Whilst plasma ghrelin concentrations are less in older than young rodents, the consequences of healthy ageing on circulating plasma ghrelin concentrations in humans are unclear. The variations in fasting ghrelin concentrations over a sixty year age range were evaluated in healthy young and older subjects (Chapter 6). Plasma ghrelin concentrations were higher in females than males, but did not correlate with age, and were inversely related to body mass index. Ghrelin was independently, and inversely, related to total body skeletal muscle mass, but not to any other body composition variable. Strategies for increasing muscle mass, through resistance exercises, may, accordingly, aid in abolishing the compensatory rise in ghrelin concentrations seen with undernutrition and weight loss. Plasma ghrelin concentrations increase before, and decrease to trough levels within one hour of ingestion of a meal. Macronutrients differ in their ability to suppress ghrelin, being earlier and more pronounced after carbohydrate, and relatively delayed after fat or protein, ingestion. The role of carbohydrate and fat digestion in the suppression of plasma ghrelin concentrations was investigated in healthy young adults (Chapter 7). The suppression of ghrelin concentrations following a sucrose drink was attenuated by acarbose, which slows small intestinal carbohydrate absorption. Ghrelin concentrations were also suppressed after consumption of a fat-enriched drink, however addition of orlistat, which reduces fat digestion and absorption, attenuated the fall in plasma ghrelin. Thus, nutrient digestion is required, in addition to exposure of the small intestine to nutrients, for suppression of ghrelin. Postprandial hypotension describes a significant fall in blood pressure occurring up to two hours after a meal. The magnitude of the fall in postprandial blood pressure depends, in part, on the macronutrient composition of a meal, and the effects are particularly discernable in older adults. Although carbohydrates are particularly potent in reducing postprandial blood pressure in older adults, fat ingestion appears to have comparable, but delayed effects. The role of fat digestion in modifying the blood pressure responses was evaluated in healthy older adults (Chapter 8). There was a fall in blood pressure after ingestion of a high-fat drink. Orlistat, a lipase inhibitor which reduces intestinal fat absorption, potentiated the fall in postprandial blood pressure after a fat-enriched drink. Gastrointestinal function and appetite can be modulated by dietary manipulation of the macronutrient composition of an individual’s diet. The intervention study described in Chapter 9 evaluated the effects of two weeks of dietary fat supplementation on the sensitivity to the satiating effects of intravenous cholecystokinin-8 in healthy older subjects. No differences were observed in fasting, or postprandial plasma cholecystokinin concentrations after the dietary supplementation period compared to regular diet. There were also no differences in spontaneous energy intake at a buffet meal in response to exogenously administered cholecystokinin between the two diet periods. Vitamin D deficiency is common, as is type 2 diabetes, and the two conditions may be linked. There is mounting evidence linking vitamin D deficiency with abnormalities of glucose and insulin metabolism. The effects of vitamin D therapy in healthy young and older adults with low vitamin D concentrations in the setting of normal or impaired glucose tolerance were evaluated (Chapter 10). Vitamin D therapy, which normalised serum 25-hydroxyvitamin D concentrations in these individuals, did not alter glucose or insulin concentrations or insulin sensitivity during an oral glucose tolerance test. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339020 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

published_or_final_version / Physiology / Master / Master of Philosophy

Peptide receptor overexpression in diseased cells and tissues, including carcinomas provides an opportunity to develop therapeutics and imaging agents that selectively bind to such cells and tissues. This dissertation presents tools and processes that can be utilized to target melanocortin and cholecystokinin receptors through multivalent binding. In Chapter 2, improved synthesis and purification methods are described for the production of Eu-chelated probes that serve to evaluate the binding efficacy of multivalent molecules through competition binding assays. Specifically, a xylenol orange-based assay for quantification of unchelated metal ions was used to determine unbound metal ion contamination and the success of metal ion removal. The use of Empore™ chelating disks was determined to be the method of choice for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates. Applying new synthesis and purification strategies, the TRF probe Eu-DTPA-PEGO-CCK4 targeted to cholecystokinin receptors was synthesized (Chapter 2) and validated via saturation and competition binding assays (Chapter 4) using a HEK293 cell line overexpressing the human cholecystokinin 2 receptor. In Chapter 3, short and efficient syntheses of multivalent molecules targeted to melanocortin receptors based on three commercially available trigonal core scaffolds, phloroglucinol, tripropargylamine, and 1,4,7-triazacyclononane, are described. These constructs were designed to further test the 24 ±5 Å inter-ligand distance suggested in recent literature for multivalent binding to melanocortin receptors. The bioactivities of these compounds were evaluated using a competitive binding assay that employed HEK293 cells engineered to overexpress the human melanocortin 4 receptor. In the course of conducting these bioassays, novel in vitro binding assay protocols were established, which led to high repeatability and robustness of the bioassays compared to previous methods. The divalent molecules exhibited 10- to 30-fold higher levels of inhibition when compared to the corresponding monovalent molecules, consistent with divalent binding. The trivalent molecules were only statistically (~2-fold) better than the divalent molecules, still consistent with divalent binding but inconsistent with trivalent binding. Possible reasons for these behaviors and planned refinements of the multivalent constructs targeting melanocortin receptors based on these scaffolds are discussed in Chapters 3 and 6.

The neuropeptides dynorphin and cholecystokinin (CCK), and their associated pronociceptive effects were investigated in the RVM. Utilizing a nerve-injury model (SNL), RT-PCR analysis revealed increases (p<0.05) of prodynorphin mRNA, and bradyinkin, B1- and B2-receptor mRNA, post-SNL, 14-days, 2-days, and 14-days, respectively. Administration of dynorphin into the RVM produced both acute and long-lasting (>30-days) tactile hypersensitivity. Administration of the B1-antagonist, DALBK and the B2-antagonist, Hoe-140, into the RVM significantly attenuated dynorphin-induced tactile hypersensitivity. Nerve-injury induced tactile hypersensitivity was significantly reversed by RVM administration of dynorphin antiserum or the B2-antagonist, Hoe-140. These data suggest that dynorphin is up-regulated in the RVM in nerve-injury, and via the activation of bradykinin receptors in the RVM, produces abnormal pain. Like dynorphin, CCK is up-regulated in the RVM in nerve-injury, with studies suggesting that elevated levels of CCK in the RVM mediate pronociceptive activity through CCK2 receptor activation, resulting in enhanced spinal nociceptive transmission. At present, it is unknown what key neurotransmitters are mediating this RVM CCK-driven effect at the level of the spinal cord. Here, spinal cerebrospinal fluid (CSF) levels of serotonin (5-HT) and prostaglandin E2 (PGE2) were measured in the lumbar spinal cord in naïve rats following CCK administration into the RVM. Following RVM CCK microinjection, an approximate 5-fold increase in spinal (CSF) PGE2 levels was observed, as compared to baseline controls. PGE2 levels showed a progressive increase with peak levels observed at the 80-minute post-CCK injection timepoint, whereas 5-HT levels in the spinal CSF remained unchanged following CCK administration into the RVM. This release of PGE2 coincided with the timecourse for CCK-induced mechanical hypersensitivity. Administration of the CCK2-antagonist YM022 prior to CCK into the RVM, significantly attenuated (>50%) the release of PGE2 in the spinal cord. The non-selective COX-inhibitor naproxen and the 5-HT3 antagonist ondansetron, both administered intrathecally, significantly attenuated RVM CCK-induced hindpaw tactile hypersensitivity. In summary, these data suggest a bradykinin- or CCK2-receptor antagonist could be used alone or in conjunction with current therapies in the treatment of chronic pain.

Sustained administration of morphine in humans and in animals induces a state of abnormal pain (i.e., hyperalgesia) which may be associated with the development of reduced analgesic efficacy (i.e., tolerance). Evidence suggests that opiate treatment may upregulate cholecystokinin (CCK), a pronociceptive peptide, in the brain and spinal cord. Therefore, we hypothesized that CCK may be upregulated by opiate treatment in the rostral ventromedial medulla (RVM) and to subsequently drive descending facilitation mechanisms to elicit hyperalgesia and antinociceptive tolerance in rats.CCK administered into the RVM of naive rats elicited hyperalgesia which was blocked by either RVM CCK2 receptor antagonist L365,260; or by bilateral lesion of dorsolateral funiculus, a major bulbospinal descending pain modulation pathway from the RVM to spinal cord.Sustained subcutaneous morphine induced hyperalgesia and spinal antinociceptive tolerance. Both effects were reversed by RVM CCK2 antagonist, suggesting that the up-regulation of the endogenous RVM CCK system played a critical role in the expression of these phenomena.Lesion of cells in the RVM which selectively express CCK2 receptors with a saporin construct (CCK-SAP) to inhibit ribosome activity, prevented morphine-induced hyperalgesia and spinal antinociceptive tolerance. These findings suggest that the integrity of the RVM CCK system is required for the development of hyperalgesia and antinociceptive tolerance induced by sustained morphine.The CCK system does not seem to play a role in setting the baseline sensory thresholds in normal rats because neither RVM L365,260 nor CCK-SAP treatment altered baseline sensory thresholds in naive rats.CCK appears to be present exclusively in nerve terminals of RVM neurons in naive rats. There was no obvious change in the levels of CCK-LI, CCK2 receptor, or CCK2 receptor mRNA in the RVM after sustained morphine treatment. However, microdialysis studies showed an approximately 5-fold increase in basal CCK levels in the RVM after sustained morphine treatment.Taken together, our results support the hypothesis that increased release of CCK in the RVM is induced by sustained morphine and drives descending facilitation to mediate morphine-induced paradoxical "pain" and spinal antinociceptive tolerance.

This dissertation will examine the phenotype of pain facilitatory neurons in the rostral ventromedial medulla (RVM) and its role in neuropathic pain states. Activation of the descending facilitation pathways might be the result of plasticity in the RVM that is driven, at least in part, by the presence and activity of cholecystokinin type-2 receptors (CCK2R) mRNA expressing neurons. The expression of either opioid mu receptors (MOR) or CCK2R mRNA in the RVM was confirmed by in situ hybridization (ISH). Pretreatment with CCK8(s)-saporin resulted in a significant loss of CCK2R mRNA positive cells in the RVM, concomitant with a blockade of CCK8(s) induced hyperalgesia. The same treatment also significantly reduced the number of neurons labeled for MOR mRNA, hinting that MOR and CCK2R mRNA signals may be co-localized in some RVM cells. Consistent with these data, pretreatment with dermorphin-saporin significantly reduced the number of MOR mRNA labeled cells in the RVM, blocked RVM CCK8(s) induced hyperalgesia and reduced the number of CCK2R mRNA positive cells in the RVM. The co-localization was further confirmed by a dual label ISH approach using 35S-labeled CCK2R and Digoxigenin-labeled MOR riboprobes. Data showed that over 80% of labeled RVM neurons co-expressed both MOR and CCK2R mRNA, ~15% expressed only CCK2R mRNA, and very few cells expressed only MOR mRNA. The above findings may suggest that elimination of CCK2R mRNA expressing neurons result in removal of the driving force for descending facilitation from RVM, hereby block the development of neuropathic pain. Rats pretreated with CCK8(s)-saporin conjugates had a full reversal of thermal sensory threshold and partial reversal of tactile threshold starting at day 5 after SNL. The lesion effects of RVM CCK-SAP were evaluated by ISH. Comparing to saporin pretreated groups, CCK8(s)-saporin pretreatment significantly reduced the numbers of CCK2R mRNA labeled neurons within RVM. The data suggest that selective ablation of CCK2R mRNA expressing cells in RVM is sufficient to block the development of neuropathic pain, and thus confirm the hypothesis that CCK2R mRNA expressing cells may be an important player in descending facilitation from RVM as a mechanism of neuropathic pain.