Investigating the physiological and pharmacological effects of the gut hormone peptide YY (PYY)

The obesity epidemic is a critical and global public health burden. Drugs that safely promote weight loss are urgently needed to halt the rising prevalence of obesity and its associated complications, such as type 2 diabetes (T2D). Gut hormones are important regulators in metabolism and have therapeutic potential as treatments for obesity and T2D. The gut hormone peptide YY (PYY) is released from the intestine after a meal. Exogenous PYY3–36 suppresses food intake in both rodents and humans, including in the obese state. PYY3–36 suppresses appetite by acting on its receptor, the Y2R. Y2R is expressed in brain appetite centres but also in the afferent vagus nerve, the main neuroanatomical link carrying information from the gut to the brain. However, the relevant contribution of the afferent vagus to the overall effects of PYY3–36 is unknown. Chemogenetic activation of vagal afferent neurones results in reduced food intake (surpassing the effects of PYY) and might have altered the immune landscape of the gastrointestinal tract. To dissect the role of the Y2R expressed in the afferent vagus, we have developed a novel microsurgical technique in the mouse. Our work suggests that vagal Y2R mediates the anorectic effect of low dose and endogenous PYY3–36 and that this vagal signalling pathway regulates short-term feeding. This anorectic effect was not caused by an aversive response. In vitro calcium imaging confirmed that PYY3–36 directly activates vagal afferents. Chronic treatment of diet-induced obese (DIO) mice with a long-acting PYY3–36 analogue, Y242, did not cause a significant body weight loss. Longitudinal tracking of individual islet function using a novel imaging platform allowed to study the effect of diet and Y242 treatment. Chronic Y242 did not improve or worsen islet function in obese mice. Therefore, PYY-based treatments might not be suitable as a single agent but have potential in combination with other gut-hormones. Vagus nerve neuromodulation has shown potential as an anti-obesity therapy and the work in this thesis adds to a better understanding of vagal afferent function which will help optimise therapeutic interventions.Open Acces

Reappraising the role of the vagus nerve in GLP-1-mediated regulation of eating.

Here, we provide a focused review of the evidence for the roles of the vagus nerve in mediating the regulatory effects of peripherally and centrally produced GLP-1 on eating behaviour and energy balance. We particularly focus on recent studies which have used selective genetic, viral, and transcriptomic approaches to provide important insights into the anatomical and functional organisation of GLP-1-mediated gut-brain signalling pathways. A number of these studies have challenged canonical ideas of how GLP-1 acts in the periphery and the brain to regulate eating behaviour, with important implications for the development of pharmacological treatments for obesity

Generation and use of new tools for the characterisation of gut hormone receptors

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.MedImmune funded Ph

Gut microbiome and health : mechanistic insights

The gut microbiota is now considered as one of the key elements contributing to the regulation of host health. Virtually all our body sites are colonised by microbes suggesting different types of crosstalk with our organs. Because of the development of molecular tools and techniques (ie, metagenomic, metabolomic, lipidomic, metatranscriptomic), the complex interactions occurring between the host and the different microorganisms are progressively being deciphered. Nowadays, gut microbiota deviations are linked with many diseases including obesity, type 2 diabetes, hepatic steatosis, intestinal bowel diseases (IBDs) and several types of cancer. Thus, suggesting that various pathways involved in immunity, energy, lipid and glucose metabolism are affected. In this review, specific attention is given to provide a critical evaluation of the current understanding in this field. Numerous molecular mechanisms explaining how gut bacteria might be causally linked with the protection or the onset of diseases are discussed. We examine well-established metabolites (ie, short-chain fatty acids, bile acids, trimethylamine N-oxide) and extend this to more recently identified molecular actors (ie, endocannabinoids, bioactive lipids, phenolic-derived compounds, advanced glycation end products and enterosynes) and their specific receptors such as peroxisome proliferator-activated receptor alpha (PPAR alpha) and gamma (PPAR gamma), aryl hydrocarbon receptor (AhR), and G protein-coupled receptors (ie, GPR41, GPR43, GPR119, Takeda G protein-coupled receptor 5). Altogether, understanding the complexity and the molecular aspects linking gut microbes to health will help to set the basis for novel therapies that are already being developed.Peer reviewe

The role of leptin receptors in the endocrine pancreas and nucleus tractus solitarius

The highly controlled regulation of pancreatic hormone secretion is vital to keep the body’s glucose concentration at a constant level. Defects in the regulation of glucose levels are involved in several metabolic diseases, including type 2 diabetes and obesity. Leptin is a satiety hormone with important roles in the maintenance of body weight and glucose homeostasis. Mice that lack leptin (ob/ob) or the leptin receptor (db/db) are massively obese and have diabetes symptoms. Leptin has been demonstrated to have an effect on glucose homeostasis that is suggested to be secondary to the obesity these animals are suffering from. Currently, it is unclear how leptin regulates glucose homeostasis. Leptin mediates its effects by interaction with its leptin receptor (LepRb), which is highly expressed in the hypothalamus, and at lower levels in the periphery. Leptin’s effect on glucose homeostasis has been proposed to be mediated via its receptor expressed on pancreatic cells affecting insulin secretion. Previous animal studies have deleted the leptin receptor in pancreatic β- and α-cells using either “leaky” or inefficient Cre-drivers resulting in conflicting results on glucose homeostasis. In this study, we use a β-cell selective Ins1Cre promoter in mice to investigate the role of leptin receptor expressed on pancreatic cells effect on glucose homeostasis. Deletion of LepRb was found to have minor effects on glucose tolerance in female animals an effect that was only detected in 8 weeks old animals. No effect was observed in male animals or in females above the age of 8 weeks. It is well established that the LepRb in hypothalamus plays an important role in regulation of energy balance. However, the LepRb is expressed in several areas outside hypothalamus, such as the nucleus of the solitary tract (NTS). GLP-1-expressing neurons in this area express the LepRb and it is therefore possible that these neurons mediate an effect on energy homeostasis or glucose homeostasis. We have therefore deleted LepRb in GLP-1 expressing neurons with a proglucagon specific promoter iGluCre. In this study, we found no effect on body weight or glucose homeostasis in animals deleted for LepRb in GLP-1 expressing neurons. Hypothalamus is the brain region that plays a key role in the regulation of feeding and energy homeostasis. This area contains anorexigenic and orexigenic neurons and intermingled with these neurons are subpopulation of neurons named RIP2Cre neurons expressing insulin. Due to the neurons location in the feeding area of the brain they are most likely having a role in energy homeostasis. Previous studies have suggested that tumour suppressor LKB1 plays a role on body weight and food intake in these neurons. Therefore, we deleted LKB1 selectively in the RIP2Cre neurons but failed to see a difference in body weight.Open Acces

Topological Analysis of Metabolic Networks Integrating Co-Segregating Transcriptomes and Metabolomes in Type 2 Diabetic Rat Congenic Series

Background: The genetic regulation of metabolic phenotypes (i.e., metabotypes) in type 2 diabetes mellitus is caused by complex organ-specific cellular mechanisms contributing to impaired insulin secretion and insulin resistance. Methods: We used systematic metabotyping by 1H NMR spectroscopy and genome-wide gene expression in white adipose tissue to map molecular phenotypes to genomic blocks associated with obesity and insulin secretion in a series of rat congenic strains derived from spontaneously diabetic Goto-Kakizaki (GK) and normoglycemic Brown-Norway (BN) rats. We implemented a network biology strategy approach to visualise shortest paths between metabolites and genes significantly associated with each genomic block. Results: Despite strong genomic similarities (95-99%) among congenics, each strain exhibited specific patterns of gene expression and metabotypes, reflecting metabolic consequences of series of linked genetic polymorphisms in the congenic intervals. We subsequently used the congenic panel to map quantitative trait loci underlying specific metabotypes (mQTL) and genome-wide expression traits (eQTL). Variation in key metabolites like glucose, succinate, lactate or 3-hydroxybutyrate, and second messenger precursors like inositol was associated with several independent genomic intervals, indicating functional redundancy in these regions. To navigate through the complexity of these association networks we mapped candidate genes and metabolites onto metabolic pathways and implemented a shortest path strategy to highlight potential mechanistic links between metabolites and transcripts at colocalized mQTLs and eQTLs. Minimizing shortest path length drove prioritization of biological validations by gene silencing. Conclusions: These results underline the importance of network-based integration of multilevel systems genetics datasets to improve understanding of the genetic architecture of metabotype and transcriptomic regulations and to characterize novel functional roles for genes determining tissue-specific metabolism