Enteroendocrine cells (EECs) express key gastrointestinal (GI) hormones including cholecystokinin (CCK), gastric inhibitory peptide (GIP), peptide tyrosine tyrosine(PYY), glucagon‐like peptide‐1 (GLP‐1) and ghrelin. EECs are characterised to contain the hormones derived from one precursor protein. Of these, CCK‐cells are typically concentrated in the proximal small intestine and release CCK upon stimulation by nutrient ligands and in so doing signal to multiple tissues to co‐ordinate and optimise digestive, absorptive functions, and instil hunger or satiety. The aims of this study were to establish whether EECs co‐expressed CCK alongside other key GI peptides and to determine a paracrine role for CCK to increase FA uptake in intestinal cells. These studies utilised an eGFP‐CCK transgenic mouse model. Tissue sections frome GFP‐CCK mice were paraffin embedded and immunostained against an array of targets. Firstly, an anti‐GFP antiserum was employed to visualise eGFP‐cells along the GI tract, and duodenal sections were dual stained for anti‐GFP and an anti‐proCCK antiserum to confirm eGFP‐cells represented CCK‐cells. A series of dual immunostaining experiments ensued to probe duodenal eGFP‐cells for a range of different hormonal targets and demonstrated that a significant number of eGFP‐CCK cells contained GIP (37%), PYY (45%), proglucagon (14%) and ghrelin (50%). Further dual‐staining experiments were carried out to stain for CCK alongside PYY, GIP or ghrelin and enabled analysis of the intracellular localisation of co‐expressing peptides, which indicated that these peptides were packaged in the same and also with indistinctly separate vesicles. These data demonstrate CCK‐cells can co‐express more than one peptide and analysis of intracellular labelling indicates they may have the ability to co‐release CCK alongside other peptides. To investigate a potential paracrine‐signalling pathway for CCK a FA uptake assay was performed using a fluorescent C12‐fatty acid (FA) analogue (Bodipy‐FA) that was analysed using fluorescent activated cell sorting (FACS). Single small intestinal cells of eGFP‐CCK mice were prepared using an EDTA chemical/mechanical dissociation method. Cell samples were either non‐treated (control) or pre‐treated with a targeted compound prior to incubation with Bodipy‐FA. Treatment of cells witholeoylethanolamide, glucagon‐like peptide‐2 (GLP‐2) or CCK increased FA uptake 2 to3‐fold and this increase was demonstrated to be carrier‐mediated. Experiments ensued employing CCK‐cell ligands to implicate activity of CCK‐cells in this process. Bombesin and L‐amino acids induced a dynamic increase in FA uptake comparable to that achieved by pre‐treatment with CCK. However, implementation of the protocol using cells from a CCK KO model achieved replicate data and therefore demonstrated these effects were not exclusive to CCK‐cells. In conclusion, data presented in this thesis establish that a spectrum of key gut hormones is expressed in individual EECs. Furthermore, a paracrine action of CCK signalling is implicated to increase the absorptive ability of neighbouring enterocytes. These data suggest that CCK‐cells have the ability to integrate nutrient signals and secrete a cocktail of hormones in response. These findings imply an increased complexity to the enteroendocrine system whereby GI peptides may work together to potentiate a desired response without requirement of signals from higher centres.

Thesis (Ph. D.)--Case Western Reserve University, 2008. / [School of Medicine] Department of Neurosciences. Includes bibliographical references.

Introduction: Appetite is often impaired in patients with gastrointestinal inflammation. Up to 75% of hospitalised Crohn's disease (CD) patients are malnourished. Recent animal research has suggested that immune mediated upregulation of enteroendocrine cell (EEC) activity plays a mechanistic role in the appetite and feeding disturbance observed during gut inflammation. The role of EEC in producing factors regulating satiety and intestinal growth is well recognised but work on their use as therapeutic targets or agents in inflammatory bowel disease (IBD) is still in its infancy. EEC peptide dynamics are further controlled through dipeptidyl peptidase (DP4) protease metabolism but no data are yet available on its expression in IBD. My aim is to understand the roles of EEC in appetite control and the maintenance of gut mucosal integrity in intestinal inflammation. Methodology: Patients with CD and healthy controls were studied. Symptoms were assessed using visual analogue scores (VAS). Gut hormone responses to a test meal were studied using a multiplex-ELISA technique, and correlated to symptoms. At the tissue level, EEC markers and transcription factors were quantified using immunohistochemistry, quantitative polymerase chain reaction (qPCR) and western blotting techniques. The same techniques were used to study DP4 expression. The effects of glucagon-like peptide-2 (GLP-2) on a gut model of the epithelial barrier were studied by measuring the transepithelial electrical resistance (TEER) across GLP-2 exposed Caco-2 cell monolayers after cytokine exposure. Tight junction protein expression in naïve and GLP-2 exposed cells was quantified by western blotting. Main Results: CD patients with active inflammation displayed a significant reduction in appetite. At the tissue level, GLP-1 and chromogranin A (CgA) were significantly upregulated. At the mRNA level significant increased expression was noted for CgA, glucagon-like peptide-1 (GLP-1), ubiquitination factor 4a and neurogenin 3. At the plasma level, total polypeptide YY (PYY) was increased. A significant correlation was seen between postprandial PYY responses and symptoms of nausea and bloating. Ghrelin, was 3-fold higher in the CD group compared to controls, and showed a reversed postprandial response with a significant correlation with the CD activity index (CDAI). Protein DP4 expression was significantly decreased at the tissue and plasma level in CD. GLP-2 increased tight junction protein expression in Caco-2 cells and maintained stable TEER and tight junction protein expression after cytokine exposure. Conclusions: The data presented are compatible with a potential role of EEC in appetite dysregulation in intestinal inflammation. An enhanced EEC response to food intake may directly affect appetite in such patients through increased gut-brain signalling. These may present tractable therapeutic targets. The decrease in mucosal DP4 expression in CD may make bioactive GLP-2 more available in the affected gut, hence improving gut mucosal integrity in intestinal inflammation. This pilot work has shown that GLP-2 has a role in maintaining gut mucosal integrity in intestinal inflammation through a positive effect on tight junction protein expression.

No description available.

Enteroendocrine hormones released from the intestine following food intake have several roles in the control of metabolism, some of which are exploited therapeutically for the treatment of type 2 diabetes. Within this thesis, focus has been on the receptors of the gut hormones glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2). In recent years there has been a surge of interest in the enteroendocrine hormones particularly due to the success of GLP-1 mimetics in the treatment of type 2 diabetes. GLP-1 is an incretin hormone, which enhances glucose induced insulin secretion by binding GLP-1 receptors (GLP1R) on pancreatic β-cells. Despite the therapeutic success, several extra-pancreatic clinical effects of GLP-1 remain unexplained. Here, a GLP1R monoclonal antagonistic antibody that can block GLP1R signalling in vivo has been developed and characterised, providing a new tool for the study of GLP1R physiology. GIP is the second incretin hormone, initially referred to as the 'ugly duckling' incretin hormone due to it's ineffectiveness in inducing insulin secretion in type 2 diabetic patients. Aside from the incretin actions, GIP is thought to be involved in the regulation of high-fat diet (HFD) induced obesity. A new transgenic mouse model expressing a fluorescent reporter under the control of the Gipr promoter has been used here to identify GIPR expressing cells. This model showed GIPR expression in the pancreas, adipose tissue, duodenum and nodose ganglia. Surprisingly GIPR expressing cells were found centrally, in areas of the hypothalamus involved in the regulation of food intake and energy expenditure. We consequently sought to investigate the function of GIPR expressing hypothalamic cells. GLP-2, unlike GLP-1 and GIP, is not an incretin hormone. Rather, GLP-2 has been implicated in the regulation of epithelial cell proliferation and apoptosis within the intestine. Therapeutically, an analogue of GLP-2 is used for the treatment of short bowel syndrome. A common missense mutation in the GLP-2 receptor (GLP2R), D470N, has been found to be associated with type 2 diabetes, and here we sought to understand the mechanism underlying this association. The D470N mutant has decreased β-arrestin recruitment, though the significance of this finding will need further research. Overall; the new monoclonal antagonistic GLP1R antibody will help to further understand GLP1R physiology, the new transgenic GIPR mouse model has contributed to the understanding of GIPR localisation, and cell based assays have identified functional implications of a polymorphism in the GLP2R associated with an increased risk of diabetes. It is hoped that further understanding of the physiology of these gut hormone receptors will be critical in the development of new therapeutics for diabetes and obesity.

The mucosa of the small intestine is clearly able to discriminate specific chemical components of ingested meals to stimulate gastrointestinal feedback pathways and reduce further food intake. Luminal carbohydrates delay gastric emptying and initiate satiation, which are mediated by reflexes via the vagus nerve upon activation of vagal afferent endings in the mucosa. Nutrients activate these nerve fibres through intermediary epithelial cells, which release neuromediators upon transduction of luminal signals through the apical membrane. 5-hydroxytryptamine (5-HT) and glucagon-like peptide-1 (GLP-1) are released from enteroendocrine cells in response to luminal carbohydrates and both slow gastric emptying and inhibit food intake via vagal afferent pathways. The molecular mechanisms for carbohydrate detection and transduction leading to 5-HT and GLP-1 release are unknown. However molecules key to transduction of taste by receptor cells in the lingual epithelium are expressed in the gastrointestinal mucosa. The studies in this thesis aimed to investigate 1) the possibility that taste molecules expressed in the intestine form part of the carbohydrate sensing pathway that leads to 5-HT and GLP-1 release, which in turn activate mucosal vagal afferents and 2) to gauge any alterations in taste molecule expression that may relate to adaptation of carbohydrate-induced gastric motility reflexes that occurs in dietary and disease states. Firstly these studies show key taste molecules, including sweet taste receptors T1R2 and T1R3, the Gprotein gustducin (alpha-subunit Gαgust), and the taste transduction channel TRPM5, are expressed in the mouse gastrointestinal mucosa shown by RT-PCR and were further localised to individual epithelial ‘taste’ cells using immunohistochemistry. Quantification of transcript levels by real time RT-PCR revealed the proximal small intestine as the preferential site of sweet taste receptor expression along the gastrointestinal tract. This finding was also confirmed in humans using gastric and intestinal mucosal biopsies obtained at enteroscopy with significantly higher transcript expression levels in the small intestine compared to stomach. In the mouse, double label immunohistochemistry with Gα[subscript]gust antibody, as a marker of intestinal taste cells, was performed using lectin UEA-1, a marker of intestinal brush cells, and 5-HT or GLP-1 to link intestinal taste transduction to 5-HT and GLP-1 release. Results show Gα[subscript]gust is expressed within a subset of all three cell types in the small intestine but predominantly within UEA-1-expressing cells. Although Gα[subscript]gust, 5-HT and GLP-1 are largely expressed in mutually exclusive cells, within the jejunum a portion Gαgust positive cells coexpressed 5-HT or GLP-1. This Indicates a subpopulation of intestinal taste cells may be dedicated to carbohydrate-evoked gastrointestinal reflexes through 5-HT and GLP-1 mediated pathways, however, taste transduction within the small intestine appears to predominantly link to alternate mediators. After nutrient detection at the luminal surface, activation of mucosal afferents by 5-HT released from enterochromaffin cells is well documented, however although vagal afferents express GLP-1 receptors direct activation has not been demonstrated. For this purpose the effects of GLP-1 on gastrointestinal vagal afferents were investigated through single fibre recordings in in vitro tissue preparations. GLP-1 had no effect on the activity of mouse gastroesophageal vagal afferents but a rat duodenal preparation proved too problematic to be able to test GLP-1 specifically on duodenal vagal afferents. Altered gastric motility in response to carbohydrate meals due to prior dietary patterns and diabetes mellitus suggest adaptation in feedback mechanisms. Towards the second aim of this thesis taste molecule expression was quantified in fed and fasted mice by real time RT-PCR and revealed taste gene transcription is altered with the changing luminal environment, specifically transcription of taste genes was significantly decreased after feeding compared to the fasted state. Studies comparing expression in the duodenum of type 2 diabetics and non-diabetic controls show no significant difference in taste transcript levels between the two groups. However taste molecule expression was correlated to blood glucose levels in diabetics suggesting transcription of these signal molecules is adapted to both luminal and systemic carbohydrate levels. Findings in both the mouse and human gastrointestinal tract in terms of intestinal chemosensing are discussed. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1363582 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 2009

The mucosa of the small intestine is clearly able to discriminate specific chemical components of ingested meals to stimulate gastrointestinal feedback pathways and reduce further food intake. Luminal carbohydrates delay gastric emptying and initiate satiation, which are mediated by reflexes via the vagus nerve upon activation of vagal afferent endings in the mucosa. Nutrients activate these nerve fibres through intermediary epithelial cells, which release neuromediators upon transduction of luminal signals through the apical membrane. 5-hydroxytryptamine (5-HT) and glucagon-like peptide-1 (GLP-1) are released from enteroendocrine cells in response to luminal carbohydrates and both slow gastric emptying and inhibit food intake via vagal afferent pathways. The molecular mechanisms for carbohydrate detection and transduction leading to 5-HT and GLP-1 release are unknown. However molecules key to transduction of taste by receptor cells in the lingual epithelium are expressed in the gastrointestinal mucosa. The studies in this thesis aimed to investigate 1) the possibility that taste molecules expressed in the intestine form part of the carbohydrate sensing pathway that leads to 5-HT and GLP-1 release, which in turn activate mucosal vagal afferents and 2) to gauge any alterations in taste molecule expression that may relate to adaptation of carbohydrate-induced gastric motility reflexes that occurs in dietary and disease states. Firstly these studies show key taste molecules, including sweet taste receptors T1R2 and T1R3, the Gprotein gustducin (alpha-subunit Gαgust), and the taste transduction channel TRPM5, are expressed in the mouse gastrointestinal mucosa shown by RT-PCR and were further localised to individual epithelial ‘taste’ cells using immunohistochemistry. Quantification of transcript levels by real time RT-PCR revealed the proximal small intestine as the preferential site of sweet taste receptor expression along the gastrointestinal tract. This finding was also confirmed in humans using gastric and intestinal mucosal biopsies obtained at enteroscopy with significantly higher transcript expression levels in the small intestine compared to stomach. In the mouse, double label immunohistochemistry with Gα[subscript]gust antibody, as a marker of intestinal taste cells, was performed using lectin UEA-1, a marker of intestinal brush cells, and 5-HT or GLP-1 to link intestinal taste transduction to 5-HT and GLP-1 release. Results show Gα[subscript]gust is expressed within a subset of all three cell types in the small intestine but predominantly within UEA-1-expressing cells. Although Gα[subscript]gust, 5-HT and GLP-1 are largely expressed in mutually exclusive cells, within the jejunum a portion Gαgust positive cells coexpressed 5-HT or GLP-1. This Indicates a subpopulation of intestinal taste cells may be dedicated to carbohydrate-evoked gastrointestinal reflexes through 5-HT and GLP-1 mediated pathways, however, taste transduction within the small intestine appears to predominantly link to alternate mediators. After nutrient detection at the luminal surface, activation of mucosal afferents by 5-HT released from enterochromaffin cells is well documented, however although vagal afferents express GLP-1 receptors direct activation has not been demonstrated. For this purpose the effects of GLP-1 on gastrointestinal vagal afferents were investigated through single fibre recordings in in vitro tissue preparations. GLP-1 had no effect on the activity of mouse gastroesophageal vagal afferents but a rat duodenal preparation proved too problematic to be able to test GLP-1 specifically on duodenal vagal afferents. Altered gastric motility in response to carbohydrate meals due to prior dietary patterns and diabetes mellitus suggest adaptation in feedback mechanisms. Towards the second aim of this thesis taste molecule expression was quantified in fed and fasted mice by real time RT-PCR and revealed taste gene transcription is altered with the changing luminal environment, specifically transcription of taste genes was significantly decreased after feeding compared to the fasted state. Studies comparing expression in the duodenum of type 2 diabetics and non-diabetic controls show no significant difference in taste transcript levels between the two groups. However taste molecule expression was correlated to blood glucose levels in diabetics suggesting transcription of these signal molecules is adapted to both luminal and systemic carbohydrate levels. Findings in both the mouse and human gastrointestinal tract in terms of intestinal chemosensing are discussed. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1363582 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 2009

The incidence of obesity is on the rise globally and is associated with many comorbidities, especially type 2 diabetes mellitus (T2DM). Bariatric surgery is the most effective intervention for weight loss and reducing obesity-associated morbidity. The most common bariatric surgeries are roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG). RYGB and SG are equally efficacious at long-term reduction of weight in obese individuals and amelioriation of T2DM. Interestingly, the improvement of glucose regulation is noted before weight loss is observed. The most likely mechanism underlying glucose homeostasis after bariatric surgery is hormonal changes in the intestine. Enteroendrocrine changes favorable of an anti-diabetic profile are noted after only a few days of receiving either RYGB or SG surgery. Most consistently, elevated postprandial GLP-1, a potent regulator of appetite and glucose control, is observed in post-bariatric surgery patients. However, data is limited regarding post-prandial GLP-1 levels beyond two years after surgery. This study will address the gap in literature by assessing postprandial elevations of GLP-1 following RYGB or SG for up to five years. We will recruit obese type-2 diabetics from an outpatient bariatric surgery clinic at Boston Medical Center scheduled to receive RYGB or SG and periodically assess postprandial GLP-1 levels to determine if they remain elevated after 5 years. Additionally, we will provide evidence if there is a correlation among changes in postprandial GLP-1, weight loss, and hemoglobin A1c at five years. Our proposed study will help direct researchers to develop safer and more efficacious interventions for obesity and T2DM.

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 2 uppsatser.

The current epidemic of obesity has been linked to an increase in fat intake associated with the Western diet. Nutrient-induced stimulation of enteroendocrine cells in the small intestine leads to the release of hormones that contribute to satiety and the control of food intake. In particular, ingested fat, specifically in the form of free fatty acids, is potent activator of enteroendocrine cells in the proximal small intestine. However, the underlying signaling cascade that free fatty acids initiate in these enteroendocrine cells, which leads to secretion of satiety hormones, is not known. In general, my research is focused on identifying nutrient-responsive pathways in enteroendocrine cells involved with the release of satiety signals and using this information to begin to develop novel drug delivery strategies to reduce food intake. In general, my results revealed that activation of the fatty acid receptor GPR120 was ecessary for the linoleic acid-induced intracellular calcium rise, a necessary precursor for hormone release. Using patch clamp recording, I discovered that linoleic acid activated enteroendocrine cells by inducing membrane depolarization, a process requiring the calcium-activated, monovalent cation permeable channel TRPM5, which is activated downstream of GPR120. To validate the unexpected finding that TRPM5 was involved in fattyacid signaling, I performed experiments using bitter compounds, whose transduction pathway is known to involve TRPM5. Enteroendocrine cells express the bitter taste receptors and release cholecystokinin in response to bitter stimuli, suggesting the probable role of gut in initiation of protective behavior against ingestion of potentially harmful substances. Armed with the data on the specifics of the fatty acid transduction, I performed experiments using nanoparticles to determine their utility for delivering pharmaceuticals specifically to the enteroendocrine cells. I fabricated and characterized PLGA nanoparticles and performed intracellular uptake studies in order to optimally delivery payloads inside cells. Finally, I validated their use by using cell-based assays to determine the effects of internalized PLGA nanoparticles on ion channels and signaling pathways involved in CCK release. Taken together, this dissertation research has identified the signaling pathways (pharmacological targets) involved in fatty acid-mediated satiety hormone release and validated the potential therapeutic use of nanoparticle-mediated drug delivery for the eventual control of food intake.