OVERARCHING RESEARCH VISION: To improve understanding of the physiology and pathophysiology of gastrointestinal vagal afferent sensory signalling with the ultimate aim to provide new pharmacological targets or dietary therapeutic approaches for the treatment of diseases such as obesity and functional dyspepsia.

To date the Vagal Afferent Research Group has made significant contributions towards this vision, transforming the research field. This includes:

• pioneering studies on the phenotypic specialisation of vagal afferent endings
• elucidation of the role of G-protein coupled receptors in vagal afferent regulation of lower oesophageal sphincter function; validating them as novel targets to treat gastro-oesophageal reflux disease.
• Elucidation of the role of mechanosensitive ion channels in visceral sensory transduction with correlation to functional outcomes.
  
  More recent research focusses on the role of gastric vagal afferent signalling in the regulation of food intake with seminal observations that vagal afferent signals:
• are dampened in high fat diet (HFD)-induced obesity and remain compromised after return to a normal diet.
• are dampened in HFD-induced obesity due to disruption in transient receptor potential, vanilloid 1 (TRPV1) channel signalling.
• exhibit circadian rhythmicity to finely tune food intake to energy demand; a property that is lost in HFD-induced obesity but maintained by time-restricted feeding.
• are regulated by gastrointestinal appetite hormones in a nutritional status dependent manner. Notably, leptin effects on gastric vagal afferents switch from ‘anorexigenic’ in lean mice to ‘orexigenic’ in HFD mice.
• are exaggerated in chronic stress conditions reflective of enhanced gastrointestinal luminal sensitivity in functional dyspepsia.

The Vagal Afferent Research Group intends to build on these strong research foundations to expand understanding on how individual subtypes of gastrointestinal afferent contribute to motor and sensory function of the gut, as it applies to food intake and appetite regulation. In addition, the group plans to establish how this system adapts to normal physiological conditions, such as pregnancy, and also how this is altered in disease, such as obesity and functional dyspepsia.   

The overall aims of the Vagal Afferent Research Groups research program for the next 5 years are to establish the:

1. subtype specificity of gastrointestinal vagal afferents in motor and sensory function.
2. plasticity of gastrointestinal vagal afferents in ‘normal’ physiological conditions, such as pregnancy.
3. molecular mechanisms responsible for the modulatory effect of hormones/peptides (e.g. leptin) on gastrointestinal vagal afferent function and the changes that occur in high fat diet-induced obesity.
4. molecular mechanisms responsible for gastrointestinal vagal afferent responses to mechanical and chemical stimuli and the changes that occur to induce:
   • hyposensitivity in high fat diet-induced obesity.
   • hypersensitivity under chronic stress conditions.

CLINICAL SIGNIFICANCE: The prevalence of obesity has more than doubled in the last 30 years and it is estimated that, by 2025, ~70% of the Australian population will be overweight or obese. According to the Australian Bureau of Statistics there were 1,043 deaths in 2009 (median age 61.1yrs) for which obesity was an underlying or associated cause of death. The total annual cost of obesity in 2008 was estimated at ~$58 billion. Consequently, there is increased impetus to improve knowledge of the human biology of obesity and the biological mechanisms that drive the condition.

Functional dyspepsia is a disease in which luminal sensitivity to gastric distension and small intestinal lipid is abnormally enhanced leading to gastrointestinal symptoms including nausea, bloating and early satiety⁽¹⁾. Functional dyspepsia affects about 20% of the population, significantly impairing quality of life. There is limited research in this area, even in humans, and important questions, relating to the mechanisms driving abnormally heightened perception, have not been addressed.

Over- or under-nutrition during pregnancy can create significant health risks for the mother and her developing fetus. Studies indicate that under- or over-nutrition in pregnancy can result in a ‘programming’ effect during the vulnerable fetal developmental periods⁽²⁾. The result is a predisposition to chronic conditions in adulthood, such as type 2 diabetes mellitus, renal⁽³⁾ and cardiovascular disease⁽⁴⁾. It also increases offspring susceptibility to obesity⁽²⁾. Despite the detrimental effects of over- and under-nutrition, on offspring health, there is limited knowledge on the role of the gastrointestinal tract in food intake and nutrient absorption during pregnancy.

BACKGROUND: In animals, the acquisition of nutrients in order to maintain life requires the intake of food. As a consequence, evolution has developed a sophisticated and well integrated multilevel system to finely control energy intake. Although great advances have been made in our basic understanding of this system the finer complexities remain a mystery. We know the gut-brain axis plays an important role in the: 1) regulation of gastrointestinal motility and secretions to optimise absorption of nutrients, and 2) regulation of appetite to control meal size. However, there is inadequate knowledge of the mechanisms involved in initiating these signals, the plasticity of these mechanisms under normal physiological conditions (i.e. pregnancy) and whether changes in these mechanisms are associated with diseases such as obesity or functional dyspepsia. In the periphery, vagal afferent sensory nerves innervating the gut are ideally positioned to respond to both the volume and type of food consumed. They can be activated by mechanical stimulation, from the physical presence of food in the gut, or chemical stimulation, by gastrointestinal peptides released in response to specific nutrients. In addition, the gastrointestinal peptides and circulating hormones can modulate the response of vagal afferents to both mechanical and chemical stimuli⁽⁵⁾. Therefore, vagal afferents display a remarkable degree of adaptability in response to daily variations in food intake and nutritional status. However, extension of this adaptability to normal physiological states, including during high energy demand, such as in pregnancy, is unknown. In addition, disruption of this system can have significant health implications. Understanding the mechanisms that drive exaggerated or dampened vagal afferent signalling will lead to new diet regimes and/or pharmacotherapies for treatment of diseases such as obesity and functional dyspepsia.

METHODOLOGY: Consistent with the Vagal Afferent Research Groups research to date, a multidisciplinary approach is utilised combining molecular (e.g. quantitative RT-PCR, Western blot, laser capture microdissection), appetite hormone measurements, electrophysiology, imaging (e.g. immunohistochemistry), functional (e.g. gastric emptying, metabolic monitoring) and behavioural (e.g. open field, elevated maze, forced swim & sucrose preference tests) studies with mouse models (e.g. HFD-induced obesity, chronic unpredictable stress, genetically modified strains, pregnancy) and human tissue.

SPECIFIC PROJECTS AVAILABLE:

The projects below provide information on the research areas available for Higher Degree by Research students. These projects can be split into smaller components to accommodate 3^rd year Research Placements and Honours students.

1. Establish the subtype specificity of gastrointestinal vagal afferents in motor and sensory function.

Background: The Vagal Afferent Research Groups research on gastric vagal afferent satiety signalling in health and obesity has contributed to a paradigm shift in understanding of gut-brain signalling. For example, we have established that there are distinct subpopulations of gastric vagal afferent, namely mucosal and tension sensitive vagal afferents. However, the exact physiological role of these afferent subtypes has not been fully elucidated. For instance, there is no direct evidence for the role of gastric mucosal receptors in motility or appetite regulation. Aim: Establish the contribution of specific subtypes of gastric vagal afferent to gut function and appetite regulation. New technology is now available to determine this aim including a transgenic mouse carrying the Cre recombinase gene driven by tartrate-resistant acid phosphatase (TRAP) promotor that will express Cre only in neurons that express the transcriptional factor Fos after a stimulus. Therefore, mucosal stimuli will restrict Cre in mucosal vagal afferent neurons. Further, injection of a Cre dependent virus to stimulate/inhibit this population of vagal afferent neurons will enable determination of function. Outcomes and Significance: This research will increase fundamental knowledge of gastric vagal afferent function and identify specific subtypes of gastric vagal afferent to target for the treatment of diseases such as obesity and functional dyspepsia.

2. Establish plasticity of gastrointestinal vagal afferents in ‘normal’ physiological conditions, such as pregnancy.

Background: During pregnancy the supply of energy must be sufficient to ensure adequate growth and development of the fetus with the degree of maternal nutrition having substantial implications for fetal development and subsequent postnatal health. For example, nutrient restriction during pregnancy can have adverse metabolic consequences in the adult offspring^{(6, 7)} with increased cardiovascular and type 2 diabetes risk^{(8, 9)}.   To meet the energy demands of the growing fetus a state of positive energy balance is developed through adaptation of homeostatic processes including the regulation of food intake^(10-12). It is known that there are gross anatomical changes in the gastrointestinal tract, predominantly the small intestine, that increase the surface area and thus increase the absorptive capacity of the gut. However, the mechanisms by which the gut senses and responds to food intake involves the interplay of multiple complex pathways that, among other processes, regulate appetite and modulate absorption of nutrients. Fundamental knowledge on the plasticity of these processes during pregnancy is lacking. We propose that gastrointestinal satiety signalling is dampened during pregnancy to permit the intake of food at a time of high energy demand. Further, we hypothesise that nutrient transporter molecule expression is increased and contributes to increased nutrient absorption during pregnancy. Objectives: (i) Characterise gastrointestinal vagal afferent satiety signalling during pregnancy; and (ii) Characterise the contribution of gastrointestinal nutrient transporter molecules in nutrient absorption during pregnancy. Outcome: This project will substantially increase understanding of the plasticity of gastrointestinal vagal afferent signalling, appetite regulation and nutrient absorption during pregnancy. Significance: This work will significantly increase knowledge of the adaptive mechanisms within the GI tract necessary to meet the energy demands of the growing fetus. Further, the findings will direct future research investigating strategies to combat over- or under-nutrition of the developing fetus.

3. Establish the molecular mechanisms responsible for the modulatory effect of hormones/peptides (e.g. leptin) on gastrointestinal vagal afferent function and the changes that occur in high fat diet-induced obesity.

Background: Appetite hormones, such as leptin and ghrelin, modulate gastric vagal afferent satiety signals in a nutritional status dependent manner^e.g.(13-16). Notably, leptin effects on gastric vagal afferents switch from ‘anorexigenic’ in lean mice to ‘orexigenic’ in HFD mice⁽¹⁴⁾. Consistent with these results, it has been reported that targeted vagal afferent leptin receptor knockout increases weight gain in lean (chow-fed) mice but paradoxically decreases weight gain of mice fed a HFD compared to wildtype controls on equivalent diets⁽¹⁷⁾. Unravelling the mechanisms that drive this switch will have significant implications for the development of new targets for the treatment of obesity and will be a primary focus of this project.  Outcomes: This project will substantially increase our understanding of the mechanism of leptin-induced modulation of  gastric vagal afferent satiety signals and establish why there is a switch in the effect of leptin on gastric vagal afferents from ‘anorexigenic’ in lean mice to ‘orexigenic’ in HFD-induced obese mice. Significance: Establishing the mechanism that underlies this switch in effect of leptin is central to the development of peripheral obesity pharmacotherapies.

4. Establish the molecular mechanisms responsible for gastrointestinal vagal afferent responses to mechanical and chemical stimuli and the changes that occur to induce: a) hyposensitivity in high fat diet-induced obesity and b) hypersensitivity under chronic stress conditions.

a) The underlying mechanisms responsible for TRPV1 signalling and its disruption in HFD-induced obesity.

Background: TRPV1 has been implicated in obesity and we have established that dampened gastric vagal afferent satiety signalling in HFD-induced obesity is due to a disruption in TRPV1 signalling⁽¹⁸⁾. Endocannabinoids (ECs: e.g. anandamide (AEA)), which are endogenous ligands for TRPV1, have peripheral effects on appetite regulation⁽¹⁹⁾. To date such effects have been considered mediated by the cannabinoid receptor 1 (CB1) with overactivity of this system increasing food intake. However, there is cross-talk between CB1 and TRPV1. We propose that dampened satiety signalling in HFD-induced obesity is due to inhibition of TRPV1 channels as a consequence of overactivity of CB1 receptors in gastric vagal afferents. Objectives: (i) Characterise the gastric vagal afferents expressing TRPV1, CB1 or both; (ii) Determine the interaction between TRPV1, ECs and CB1 on gastric vagal afferent mechanosensitivity and gastric vagal afferent neurone excitability in lean and HFD-induced obese mice; (iii) Measure levels of endocannabinoids in the stomach in lean and HFD-induced obese mice; and (iv) Establish the physiological significance of TRPV1 in gastric vagal afferent satiety signalling in lean and HFD-induced obese mice. Outcome: This project will substantially increase understanding of the interactions between the EC system and TRPV1 in the control of gastric vagal afferent satiety signalling. It will also increase understanding of how these interactions are altered in obesity to dampen gastric vagal afferent satiety signalling and perpetuate the problem of obesity. Significance: This work will identify much-needed new targets for the treatment of obesity.

b) Mechanisms that drive GI vagal afferent hypersensitivity under chronic stress conditions.

Background: Functional dyspepsia is characterised by hypersensitivity to gastrointestinal stimuli relating to a meal⁽¹⁾ and is associated with symptoms including early satiety, fullness, bloating, nausea and epigastric pain. Psychological disorders (e.g. anxiety and depressive disorders) often precede or exacerbate functional dyspepsia symptoms^{(20, 21)}. We have preliminary data demonstrating chronic unpredictable stress induces hypersensitivity of gastric vagal afferents. The symptoms of functional dyspepsia are associated with both the stomach and duodenum. Using the chronic stress model along with the gastrointestinal vagal afferent preparations we aim to unravel the mechanisms responsible for vagal afferent hypersensitivity, in both the stomach and small intestine. In patients with IBS, which is predominantly a functional disorder of the lower gastrointestinal tract, the response to capsaicin (TRPV1 ligand) is potentiated compared to healthy volunteers⁽²²⁾. TRPV1 receptors are involved in vagal afferent signalling⁽¹⁸⁾ and therefore gastric vagal afferent hypersensitivity observed in chronic stress conditions could be due to heightened TRPV1 mediated signalling and, as above, alterations in the interaction between TRPV1, CB1 and endocannabinoids. Stress-induced hypersensitivity may also be mediated through inflammation since the enhanced capsaicin response, in IBS patients, is mimicked by pre-incubation with the mast cell mediator histamine and is dependent on histamine 1 receptor (H1R) activation⁽²²⁾. Objective: (i) Characterise the gastrointestinal vagal afferents expressing TRPV1, CB1 and H1R; and (ii) Determine the relationship between TRPV1 and histamine (from mast cells, gastrointestinal mucosa or microbiome) in chronic stress induced hypersensitivity. Outcome: The proposed research will significantly advance understanding of the mechanisms involved in chronic stress-induced vagal afferent hypersensitivity.  Significance: This research will provide new pharmacological targets or diet regimes for the treatment of functional dyspepsia patients with psychological stress-associated symptoms.

REFERENCES

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15.       Kentish SJ, Ratcliff K, Li H, Wittert GA, Page AJ. High fat diet induced changes in gastric vagal afferent response to adiponectin. Physiol Behav. 2015 Dec 1;152(Pt B):354-62. DOI: 10.1016/j.physbeh.2015.06.016.

16.       Kentish SJ, Li H, Frisby CL, Page AJ. Nesfatin-1 modulates murine gastric vagal afferent mechanosensitivity in a nutritional state dependent manner. Peptides. 2017 Mar;89:35-41. DOI: 10.1016/j.peptides.2017.01.005.

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