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Nutrient sensing in the GI tract
The gastrointestinal (GI) tract plays an important role in the regulation of energy homeostasis. It is the initial interface of the body at which nutrients are digested, absorbed and assimilated. EECs are specialized chemosensing epithelial cells dispersed along the mucosa of the GI tract. EECs are the first level of integration of information from the gut lumen that respond to the presence or absence of nutrients and secrete a variety of gut-derived hormones such as CCK. They are strategically located in the mucosa of the GI tract to taste and sense luminal contents and release hormones basolaterally to enter into the circulation (Figure 1.1). Gut-derived hormones either act directly on central receptors or they bind to their receptors found on vagus nerve fiber terminals to activate vago vagal pathway. In turn, efferent signals will target peripheral targets to control gut functions and ingestive responses.
Recent advances in understanding nutrient sensing
ECCs are chemosensing cells that finely coordinate nutrient sensing with metabolic and behavioral functions such as regulating energy homeostasis and glucose metabolism. ECCs release hormones such as CCK, glucagon-like-peptide 1 (GLP-1) and peptide YY (PYY) in response to nutrient ingestion (6). These signals are important in regulating appetite and body weight; in a prepandial state, circulating levels of orexigenic peptides are high to stimulate food intake and conversely, in postprandial states, anorexigenic peptides are secreted to inhibit food intake.
EECs are located in the mucosa of the gastrointestinal epithelium to taste and sense nutrients passing through the lumen. Peptides known to regulate food intake are released on the basolateral side and act either on nearby neural pathways or can enter directly into the circulation. Schematic taken from Cummings D.E and Overduin J, 2007 (44).
EECs are scattered throughout the epithelium of the GI tract and represent less than 1% of the epithelial cell population making the study of native EECs difficult. However, the development of transgenic mice expressing green fluorescent protein (GFP) under the control of specific EECs gene promotors has greatly enhanced the ability to study gut EECs in cell culture. GFP is used as a marker of gene expression and protein localization in living organisms without the use of an exogenous substrate. GFP with enteroendocrine promoters label hormone precursors, chemosensor receptors and granular proteins.
Given that EECs are difficult to study, much of what we know about EECs and nutrient sensing stems from cell lines such as STC-1 and GLUTag cells. These cells have been helpful in understanding intracellular signaling, however, they are transformed cells whereby their exact protein content or responses to stimuli may not replicate in vivo EEC function. Physiological and anatomical characterization of ECCs has been greatly enhanced through GFP-tagged mice. Traditionally, ECCs have been classified according to hormonal content, morphology and localization in the GI tract leading to the concept of “one cell type, one hormone”. However, results from transgenic mice with tagged EECs have questioned this concept. GFP-tagged mice with enteroendocrine promoters have demonstrated that multiple hormones are synthesized and released from a single ECC revealing the pluritrophic actions of EECs (7, 8). For example, studies using CCK-GFP mice have demonstrated that CCK is coexpressed with GLP-1, glucose-dependent insulinotrophic peptide (GIP), PYY, secretin and neurotensin (9, 10). Surprisingly, Egeord et al. found that promoters for CCK and GLP-1 were scattered throughout the gut rather than segregated in either the proximal or distal gut (8). The hormone content of individual cells differs according to their localization. For example, EECs from the proximal gut contain high levels of CCK and low levels of GLP-1. Conversely, EECs cell from the distal gut have high levels of GLP-1 and low levels of CCK (9). An extensive study on CCK-containing cells demonstrated that the coexpression of hormones, such as CCK and ghrelin, was consistent between immature and fully differentiated cells (10).
The anatomical arrangement within each cell has been extensively studied in the last few years; PYY and GLP-1 secretory granules have been demonstrated to be contained at the base of L-cells. Furthermore L cells contain a prominent basal cytoplasmic process (7, 11). GLP-1 and PYY granules are contained in separate storage organelles within the same cell and these organelles can be selectively mobilized according to different stimuli (12).
Gastrointestinal functions are modulated by changes in luminal contents to protect against harmful substances. EECs act as primary chemoreceptors by releasing signaling peptides in response to changes in the luminal environment. Transgenic GFP-mice has been possible to look at the mechanism of nutrient sensing directly on native EECs. For example, using duodenal extract from CCK-GFP mice, it has been demonstrated that amino acid-induced CCK release acts through calcium sensing receptor (12). There are several molecular responses stimulated by nutrient sensing such as protein transport, glucose sensors and G–protein coupled receptors (GPCR). Convincing evidence suggests that EECs in the GI tract express several G-protein coupled receptors that are involved in chemosensing (13). These receptors are stimulated by glucose, calcium and short and long chain fatty acids. Many gut-derived proteins have been shown to act specifically through GPCR. It has been established that GPCR are involved in nutrient sensing of fatty acid (12, 13). In mouse intestine, GPR120 is colocalized with GLP-1(13). Furthermore, fatty acid induced secretion of GLP-1 is attenuated in GPR120 knockout mice (13). GPR40 gene and protein expression was identified in CCK-expressing ECCs. GPR40 was shown to directly mediate fatty acid-induced CCK secretion, which was further attenuated by the deletion of GPR40 (14).
Importance of gut-derived hormones on regulation of food intake
Gut-derived hormones are secreted in the absence or presence of nutrients to control food intake. They are localized in the epithelium of the GI tract in an ideal location to respond to luminal contents. Their patterns of release alter according to nutrient availability in ways that could affect short and long-term feeding behavior. Studies using exogenous administrations of gut peptides have revealed the functional importance of signals that arise from the GI tract in regulating energy homeostasis.
Glucagon like peptide-1
GLP-1 is derived from the expression of the transcriptional product of the pre-proglucagon gene in intestinal L-cells and pancreatic α-cells. GLP-1 is released by L-cells predominately found in the distal gut in response to nutrient ingestion. Glucose and fat are the most potent stimulators of GLP-1 stimulation (15). GLP-1 receptors (GLP-1R) are found throughout the peripheral and CNS, where its highest expression is found in the lung, distal gut and brain (16, 17). GLP-1 was considered until recently for its role as an incretin hormone and ability to restore glucose homeostasis in type 2 diabetic patients. In fact, GLP-1 analogs and DPP-IV inhibitors are on the market for the treatment of type 2 diabetes. Emerging evidence suggest a possible physiological role for GLP-1 in regulating food intake. Exogenous administration of GLP-1 or its long-acting analogs dose-dependently inhibit food consumption and the satiating effects of GLP-1 are attenuated by prior administration of GLP-1R antagonist (18, 19). Given that GLP-1Rs are found in the peripheral and CNS, the site of action of native GLP-1 in regulating food intake is unclear and this is discussed in further detail in the GLP-1 and control of food intake section.
GLP-1 has specifically been implicated in the pathogenesis of obesity. DIO leads to blunted GLP-1 release; decreased postprandial plasma concentrations and blunted anorexigenic response to GLP-1R activation (20, 21). Duca et al. have demonstrated that DIO results in impaired GLP-1 satiation signaling; DIO rats have an attenuated response to GLP-1R antagonist exendin 4 (Ex-4) compared to controls. Furthermore, DIO rats have decreased GLP-1 protein expression in the intestinal epithelium indicating a reduction in intestinal nutrient response from L-cells (22). Evidence suggests that GLP-1 would be an effective treatment for obesity. Plasma concentrations of GLP-1 dramatically increase in patients following bariatric surgery (23). Infusions of GLP-1 will induce satiation in lean adults by suppressing food intake and meal size. Interestingly, this effect is preserved in obese subjects implying GLP-1 as a prime candidate for anti-obesity treatments (24).
Ghrelin
The discovery of ghrelin opened a new frontier to research in energy homeostasis in that ghrelin is currently the only circulating hormone that can stimulate food intake and adiposity in humans and rodents in comparison to the large family of identified anorexigenic gut hormones. Ghrelin is a 28-amino acid polypeptide produced mainly by endocrine X/A-like cells in rodents and P/D1 in humans located in the gastric epithelium (25). Although the stomach is the main site of secretion, ghrelin is also secreted by the pituitary, hypothalamus, lung, heart and pancreas. Acetylated ghrelin, which is converted by ghrelin O-acyltransferase (GOAT) enzyme, acts on the growth hormone secretatgogue receptor (GHS-R) which is constitutively expressed (26, 27).
GHS-R is predominately found in the pituitary and the hypothalamus (28). The biological functions of ghrelin are widespread; it plays a role in lipid metabolism, glucose homeostasis and growth hormone release (28, 29). Additionally, ghrelin stimulates appetite, body weight and adiposity. Interestingly, circulating ghrelin concentrations are decreased in obese individuals compared to their lean controls (30). Given that ghrelin plasma levels are negatively correlated with insulin and leptin plasma levels, the downregulation in ghrelin may be a consequence of elevated insulin or leptin. However, antagonizing ghrelin receptor can decrease food intake in obese Zucker rats (31) possibly blocking the constitutive receptor activity (27). Given that ghrelin is involved in meal initiation makes it an attractive target in understanding feeding and energy homeostasis.
Leptin
Leptin is a 167-amino acid peptide that is mainly expressed in white adipose tissue but also found in a variety of tissues such as placenta, stomach, skeletal muscle and pituitary glands. Leptin is produced and secreted predominately by adipose tissue and the stomach (32). Circulating leptin levels positively correlate with body adiposity which reflects long term energy stores (33). Leptin is a gut and adipose tissue-derived hormone that regulates a range of biological functions and processes; including energy intake and expenditure, body fat, neuroendocrine systems, autonomic function, and insulin and glucose balance (34) (Figure 1.2). It exerts its effect by binding to its receptor, LepR, which is located throughout the CNS (35). The highest expression of the long isoform of LepR is found in the hypothalamus, an area known to regulate energy homeostasis. Six isoforms of LepR have been identified in rats; the long form has been implicated in the suppression of food intake and stimulation of energy expenditure (34).
The presence of leptin was predicted before it was cloned in 1994. Coleman et al. performed parabiosis studies in which they joined ob/ob mice, those lacking leptin, and db/db mice, those lacking leptin receptor. They concluded that ob/ob mice were missing a circulating factor that was abundant in db/db mice. The factor could cure obesity in ob/ob mice but not in db/db mice (36). The discovery of the leptin was initially thought as a cure for human obesity however, daily injections of recombinant leptin only fully corrected obesity in few cases of leptin deficient patients. It was discovered that a majority of the obese population have high circulating leptin levels and are unresponsive to leptin thereby defining them as leptin resistant (36). Evidence has undoubtedly highlighted the importance of leptin signaling for the maintenance of energy homeostasis.
Obese individuals have high circulating leptin levels which fail to reduce energy intake (33). Moreover, the response to central administrations of leptin is attenuated in mice in part due to the development of leptin resistance in the hypothalamus (37). The importance of leptin in energy homeostasis is most evident in leptin deficient models. Ob/ob mice with global leptin deficiency exhibit hyperphagia, low metabolic rate and rapid onset of obesity, associated with high expression of orexigenic neuropeptide Y (NPY) and melanin-concentrating hormone (MCH) and low expression of anorexigenic pro-opiomelanocortin (POMC) in the hypothalamus (34, 38).
Cholecystokinin
CCK was the first identified gut-derived hormone shown to have satiating effects on ingestive behavior. CCK is secreted predominately from I cells found in the upper small intestine and is released in response to fat. Plasma levels markedly increase in response to a meal. There are two subtypes of CCK receptors, CCK1R and CCK2R. CCK binds to its receptors to induce gallbladder contraction, delayed gastric emptying and release of pancreatic enzyme to induce digestion (Figure 1.3). CCK1Rs are predominately responsible for mediating CCK-induced effects on short-term food intake (39, 40). CCK is involved in the regulation of food intake and GI function.
CCK inhibits food intake in a dose dependent manner in many species including humans (41, 42). Central and peripheral administrations induce inhibitory effects on energy intake. Specifically, exogenous CCK inhibits cumulative food intake by reducing meal size and increasing intermeal interval in rodents and humans (39, 43). Perfusions of fat or protein into the small intestine inhibit feeding and the administration of CCK1R antagonists will reverse these effects (41). The satiating actions of CCK regulate short-term feeding; CCK1R knockout (KO) mice have no difference in body weight and cumulative food intake compared to wildtype (WT) mice, however, their early meal events are altered. For example, CCK1R KO mice ingest larger, longer first compared to their control littermates (44).
The major site of action of CCK is the vague nerve; damage to the vagus nerve attenuates the satiating effects of CCK (41). Exogenous administrations of CCK to rats will rapidly induce vagal electrophysiological activity (45). CCK binds to CCK1R present on the vagus nerve to terminate feeding and induce satiety integrated by the NTS, the area where vagal afferents terminate (39). CCK has been demonstrated as the master regulator of VAN phenotype switch, which alters sensitivity of VAN to fasting and refeeding conditions. This mechanism is further elucidated in the Phenotypic Changes of VAN According to Feeding Status section.
Schematic representing that CCK has various biological activities; it delays gastric emptying, increases pancreatic enzymes to facilitate digestion and induces bile secretion from gall bladder.
Table of contents :
Chapter 1: Literature Review on gut-derived peptides and their mechanism of action on vagal afferent neurons
1.1 Introduction
1.2 Nutrient sensing in the GI tract
1.2.1 Recent advances in understanding nutrient sensing
1.3 Importance of gut-derived hormones on regulation of food intake
1.3.1 Glucagon like peptide-1
1.3.2 Ghrelin
1.3.3 Leptin
1.3.4 Cholecystokinin
1.4 The Vagus Nerve
1.4.1 Phenotypic Changes of VAN According to Feeding Status
1.4.2 VAN phenotype in diet induced obesity
1.4.3 Summary
1.5 GLP-1 secretion in the GI tract
1.6 The insulinotropic activity of GLP-1
1.7 GLP-1 and the control of food intake
1.7.1 Evidence that Ghrelin Modulates GLP-1-induced Actions
1.7.2 Evidence that Leptin Interacts with GLP-1 actions
1.7.3 Summary
1.8 The role of leptin in obesity
1.8.1 Summary
1.9 Sex differences influence energy homeostasis
1.10 Conclusion
1.11 Objectives of Dissertation
1.12 References
Chapter 2: Experimental Chapter on investigating the mechanism of action of gut-derived peptides on vagal afferent neurons
2.1: Ability of Glucagon like Peptide-1 to Decrease Food Intake is Dependent on Nutritional Status
2.1.1 Abstract
2.1.2 Highlights
2.1.3 Introduction
2.1.4 Methods
2.1.5 Results
2.1.6 Discussion
2.1.7 References
.2: Ghrelin inhibits translocation of Glucagon like Peptide-1 Receptors to the plasma membrane of vagal afferent neurons
2.2.1 Abstract
2.2.2 Highlights
2.2.3 Introduction
2.2.4 Methods
2.2.5 Results
2.2.6 Discussion
2.2.7 References
3: Deletion of leptin signaling in vagal afferent neurons results in hyperphagia and obesity
2.3.1 Abstract
2.3.2 Highlights
2.3.3 Introduction
2.3.4 Methods
2.3.5 Results
2.3.6 Discussion
2.3.7 References
4: Knockdown of Leptin Receptor on vagal afferent neurons drives obesity differently in female than male mice
2.4.1 Abstract
2.4.2 Highlights
2.4.3 Introduction
2.4.4 Methods
2.4.5 Results
2.4.6 Discussion
2.4.7 References
Chapter 3: Discussion and Conclusion
3.1 Introduction
3.2 Objectives and aims
3.3 Summary of findings
3.4 Main findings
3.5 Limitations
3.6 Possible future research
3.7 Conclusion
3.8 References
