Alterations in GLP-1 and PYY release with aging and body mass in the human gut.

The lining of our intestinal surface contains an array of hormone-producing cells that are collectively our bodies' largest endocrine cell reservoir. These "enteroendocrine" (EE) cells reside amongst the billions of absorptive epithelial and other cell types that line our gastrointestinal tract and can sense and respond to the ever-changing internal environment in our gut. EE cells release an array of important signalling molecules that can act as hormones, including glucagon-like peptide (GLP-1) and peptide YY (PYY) which are co-secreted from L cells. While much is known about the effects of these hormones on metabolism, insulin secretion and food intake, less is understood about their secretion from human intestinal tissue. In this study we assess whether GLP-1 and PYY release differs across human small and large intestinal tissue locations within the gastrointestinal tract, and/or by sex, body weight and the age of an individual. We identify that the release of both hormones is greater in more distal regions of the human colon, but is not different between sexes. We observe a negative correlation of GLP-1 and BMI in the small, but not large, intestine. Increased aging correlates with declining secretion of both GLP-1 and PYY in human large, but not small, intestine. When the data for large intestine is isolated by region, this relationship with age remains significant for GLP-1 in the ascending and descending colon and in the descending colon for PYY. This is the first demonstration that site-specific differences in GLP-1 and PYY release occur in human gut, as do site-specific relationships of L cell secretion with aging and body mass.

The Impact of Long-Term Macrolide Exposure on the Gut Microbiome and Its Implications for Metabolic Control.

Long-term low-dose macrolide therapy is now widely used in the treatment of chronic respiratory diseases for its immune-modulating effects, although the antimicrobial properties of macrolides can also have collateral impacts on the gut microbiome. We investigated whether such treatment altered intestinal commensal microbiology and whether any such changes affected systemic immune and metabolic regulation. In healthy adults exposed to 4 weeks of low-dose erythromycin or azithromycin, as used clinically, we observed consistent shifts in gut microbiome composition, with a reduction in microbial capacity related to carbohydrate metabolism and short-chain fatty acid biosynthesis. These changes were accompanied by alterations in systemic biomarkers relating to immune (interleukin 5 [IL-5], IL-10, monocyte chemoattractant protein 1 [MCP-1]) and metabolic (serotonin [5-HT], C-peptide) homeostasis. Transplantation of erythromycin-exposed murine microbiota into germ-free mice demonstrated that changes in metabolic homeostasis and gastrointestinal motility, but not systemic immune regulation, resulted from changes in intestinal microbiology caused by macrolide treatment. Our findings highlight the potential for long-term low-dose macrolide therapy to influence host physiology via alteration of the gut microbiome. IMPORTANCE Long-term macrolide therapy is widely used in chronic respiratory diseases although its antibacterial activity can also affect the gut microbiota, a key regulator of host physiology. Macrolide-associated studies on the gut microbiota have been limited to short antibiotic courses and have not examined its consequences for host immune and metabolic regulation. This study revealed that long-term macrolides depleted keystone bacteria and impacted host regulation, mediated directly by macrolide activity or indirectly by alterations to the gut microbiota. Understanding these macrolide-associated mechanisms will contribute to identifying the risk of long-term exposure and highlights the importance of targeted therapy for maintenance of the gut microbiota.

Role of 5-HT in the enteric nervous system and enteroendocrine cells.

Since the 1950s, considerable circumstantial evidence had been presented that endogenous 5-HT (serotonin) synthesized from within the wall of the gastrointestinal (GI) tract played an important role in GI motility and transit. However, identifying the precise functional role of gut-derived 5-HT has been difficult to ascertain, for a number of reasons. Over the past decade, as recording techniques have advanced significantly and access to new genetically modified animals improved, there have been major new insights and major changes in our understanding of the functional role of endogenous 5-HT in the GI tract. Data from many different laboratories have shown that major patterns of GI motility and transit still occur with minor or no, change when all endogenous 5-HT is pharmacologically or genetically ablated from the gut. Furthermore, antagonists of 5-HT3 receptors are equally, or more potent at inhibiting GI motility in segments of intestine that are completely depleted of endogenous 5-HT. Here, the most recent findings are discussed with regard to the functional role of endogenous 5-HT in enterochromaffin cells and enteric neurons in gut motility and more broadly in some major homeostatic pathways.

The gut-brain axis: spatial relationship between spinal afferent nerves and 5-HT-containing enterochromaffin cells in mucosa of mouse colon.

Cross talk between the gastrointestinal tract and brain is of significant relevance for human health and disease. However, our understanding of how the gut and brain communicate has been limited by a lack of techniques to identify the precise spatial relationship between extrinsic nerve endings and their proximity to specific cell types that line the inner surface of the gastrointestinal tract. We used an in vivo anterograde tracing technique, previously developed in our laboratory, to selectively label single spinal afferent axons and their nerve endings in mouse colonic mucosa. The closest three-dimensional distances between spinal afferent nerve endings and axonal varicosities to enterochromaffin (EC) cells, which contain serotonin (5-hydroxytryptamine; 5-HT), were then measured. The mean distances (± standard deviation) between any varicosity along a spinal afferent axon or its nerve ending, and the nearest EC cell, were 5.7 ± 6.0 µm (median: 3.6 µm) and 26.9 ± 18.6 µm (median: 24.1 µm), respectively. Randomization of the spatial location of EC cells revealed similar results to this actual data. These distances are ∼200-1,000 times greater than those between pre- and postsynaptic membranes (15-25 nm) that underlie synaptic transmission in the vertebrate nervous system. Our findings suggest that colonic 5-HT-containing EC cells release substances to activate centrally projecting spinal afferent nerves likely via diffusion, as such signaling is unlikely to occur with the spatial fidelity of a synapse.NEW & NOTEWORTHY We show an absence of close physical contact between spinal afferent nerves and 5-HT-containing EC cells in mouse colonic mucosa. Similar relative distances were observed between randomized EC cells and spinal afferents compared with actual data. This spatial relationship suggests that substances released from colonic 5-HT-containing EC cells are unlikely to act via synaptic transmission to neighboring spinal afferents that relay sensory information from the gut lumen to the brain.

Distinguishing the contributions of neuronal and mucosal serotonin in the regulation of colonic motility.

BACKGROUND: Specialized enterochromaffin (EC) cells within the mucosal lining of the gut synthesize and secrete almost all serotonin (5-hydroxytryptamine, 5-HT) in the body. Significantly lower amounts of 5-HT are made by other peripheral tissues and serotonergic neurons within the enteric nervous system (ENS). EC cells are in close proximity to 5-HT receptors in the ENS, and the role of 5-HT as a modulator of gut motility, particularly colonic motor complexes, has been well defined. However, the relative contribution of neuronal 5-HT to this process under resting and stimulus-evoked conditions is unclear. METHODS: In this study, we combined the use of the selective serotonin transporter (SERT) inhibitor, fluoxetine, with two models of mucosal 5-HT depletion-surgical removal of the mucosa and our Tph1Cre/ERT2 ; Rosa26DTA mouse line-to determine the relative contribution of neuronal and mucosal 5-HT to resting and distension-evoked colonic motility. KEY RESULTS: Fluoxetine significantly reduced the frequency of colonic migrating complexes (CMCs) in flat-sheet preparations with the mucosa present and in intact control Tph1-DTA colons in which EC cells were present. No such effect was observed in mucosa-free preparations or in intact Tph1-DTA preparations lacking EC cell 5-HT. CONCLUSIONS AND INFERENCES: We demonstrate that mucosal 5-HT release plays an important role in distension-evoked colonic motility, and that SERT inhibition no longer alters gut motility when EC cells are absent, thus demonstrating that ENS 5-HT does not play a role in regulating gut motility.

The gut microbiome and mental health: advances in research and emerging priorities.

The gut microbiome exerts a considerable influence on human neurophysiology and mental health. Interactions between intestinal microbiology and host regulatory systems have now been implicated both in the development of psychiatric conditions and in the efficacy of many common therapies. With the growing acceptance of the role played by the gut microbiome in mental health outcomes, the focus of research is now beginning to shift from identifying relationships between intestinal microbiology and pathophysiology, and towards using this newfound insight to improve clinical outcomes. Here, we review recent advances in our understanding of gut microbiome-brain interactions, the mechanistic underpinnings of these relationships, and the ongoing challenge of distinguishing association and causation. We set out an overarching model of the evolution of microbiome-CNS interaction and examine how a growing knowledge of these complex systems can be used to determine disease susceptibility and reduce risk in a targeted manner.

The composition of the gut microbiota following early-life antibiotic exposure affects host health and longevity in later life.

Studies investigating whether there is a causative link between the gut microbiota and lifespan have largely been restricted to invertebrates or to mice with a reduced lifespan because of a genetic deficiency. We investigate the effect of early-life antibiotic exposure on otherwise healthy, normal chow-fed, wild-type mice, monitoring these mice for more than 700 days in comparison with untreated control mice. We demonstrate the emergence of two different low-diversity community types, post-antibiotic microbiota (PAM) I and PAM II, following antibiotic exposure. PAM II but not PAM I mice have impaired immunity, increased insulin resistance, and evidence of increased inflammaging in later life as well as a reduced lifespan. Our data suggest that differences in the composition of the gut microbiota following antibiotic exposure differentially affect host health and longevity in later life.

Pharmacological and structure-activity relationship studies of oleoyl-lysophosphatidylinositol synthetic mimetics.

Metabolic diseases, such as obesity and type 2 diabetes, are relentlessly spreading worldwide. The beginning of the 21st century has seen the introduction of mechanistically novel types of drugs, aimed primarily at keeping these pathologies under control. In particular, an important family of therapeutics exploits the beneficial physiology of the gut-derived glucagon-like peptide-1 (GLP-1), with important clinical benefits, from glycaemic control to cardioprotection. Nonetheless, these protein-based drugs act systemically as exogenous GLP-1 mimetics and are not exempt from side effects. The food-derived lipid oleoyl-lysophosphatidylinositol (LPI) is a potent GPR119-dependent GLP-1 secreting agent. Here we present a structure-activity relationship (SAR) study of a synthetic library of oleoyl-LPI mimetics capable to induce the physiological release of GLP-1 from gastrointestinal enteroendocrine cells (EECs). The best lead compounds have shown potent and efficient release of GLP-1 in vitro from human and murine cells, and in vivo in diabetic db/db mice. We have also generated a molecular model of oleoyl-LPI, as well as its best performing analogues, interacting with the orthosteric site of GPR119, laying foundational evidence for their pharmacological activity.

Dynamin regulates L cell secretion in human gut.

BACKGROUND: The mechanochemical enzyme dynamin mediates endocytosis and regulates neuroendocrine cell exocytosis. Enteroendocrine L cells co-secrete the anorectic gut hormones glucagon-like peptide 1 (GLP-1) and peptide YY (PYY) postprandially and is a potential therapeutic target for metabolic diseases. In the present study, we aimed to determine if dynamin is implicated in human L cell secretion. METHODS: Western blot was performed on the murine L cell line GLUTag. Static incubation of human colonic mucosae with activators and inhibitors of dynamin was carried out. GLP-1 and PYY contents of the secretion supernatants were assayed using ELISA. RESULTS AND CONCLUSION: s: Both dynamin I and II are expressed in GLUTag cells. The dynamin activator Ryngo 1-23 evoked significant GLP-1 and PYY release from human colonic mucosae while the dynamin inhibitor Dynole 3-42 significantly inhibited release triggered by known L cell secretagogues. Thus, the cell signaling regulator dynamin is able to bi-directionally regulate L cell hormone secretion in the human gut and may represent a novel target for gastrointestinal-targeted metabolic drug development.

A Gut-Intrinsic Melanocortin Signaling Complex Augments L-Cell Secretion in Humans.

OBJECTIVE: Hypothalamic melanocortin 4 receptors (MC4R) are a key regulator of energy homeostasis. Brain-penetrant MC4R agonists have failed, as concentrations required to suppress food intake also increase blood pressure. However, peripherally located MC4R may also mediate metabolic benefits of MC4R activation. Mc4r transcript is enriched in mouse enteroendocrine L cells and peripheral administration of the endogenous MC4R agonist, α-melanocyte stimulating hormone (α-MSH), triggers the release of the anorectic hormones Glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) in mice. This study aimed to determine whether pathways linking MC4R and L-cell secretion exist in humans. DESIGN: GLP-1 and PYY levels were assessed in body mass index-matched individuals with or without loss-of-function MC4R mutations following an oral glucose tolerance test. Immunohistochemistry was performed on human intestinal sections to characterize the mucosal MC4R system. Static incubations with MC4R agonists were carried out on human intestinal epithelia, GLP-1 and PYY contents of secretion supernatants were assayed. RESULTS: Fasting PYY levels and oral glucose-induced GLP-1 secretion were reduced in humans carrying a total loss-of-function MC4R mutation. MC4R was localized to L cells and regulates GLP-1 and PYY secretion from ex vivo human intestine. α-MSH immunoreactivity in the human intestinal epithelia was predominantly localized to L cells. Glucose-sensitive mucosal pro-opiomelanocortin cells provide a local source of α-MSH that is essential for glucose-induced GLP-1 secretion in small intestine. CONCLUSION: Our findings describe a previously unidentified signaling nexus in the human gastrointestinal tract involving α-MSH release and MC4R activation on L cells in an autocrine and paracrine fashion. Outcomes from this study have direct implications for targeting mucosal MC4R to treat human metabolic disorders.

Serotonin Deficiency Is Associated With Delayed Gastric Emptying.

BACKGROUND & AIMS: Gastrointestinal (GI) motility is regulated by serotonin (5-hydroxytryptamine [5-HT]), which is primarily produced by enterochromaffin (EC) cells in the GI tract. However, the precise roles of EC cell-derived 5-HT in regulating gastric motility remain a major point of conjecture. Using a novel transgenic mouse line, we investigated the distribution of EC cells and the pathophysiologic roles of 5-HT deficiency in gastric motility in mice and humans. METHODS: We developed an inducible, EC cell-specific Tph1CreERT2/+ mouse, which was used to generate a reporter mouse line, Tph1-tdTom, and an EC cell-depleted line, Tph1-DTA. We examined EC cell distribution, morphology, and subpopulations in reporter mice. GI motility was measured in vivo and ex vivo in EC cell-depleted mice. Additionally, we evaluated 5-HT content in biopsy and plasma specimens from patients with idiopathic gastroparesis (IG). RESULTS: Tph1-tdTom mice showed EC cells that were heterogeneously distributed throughout the GI tract with the greatest abundance in the antrum and proximal colon. Two subpopulations of EC cells were identified in the gut: self-renewal cells located at the base of the crypt and mature cells observed in the villi. Tph1-DTA mice displayed delayed gastric emptying, total GI transit, and colonic transit. These gut motility alterations were reversed by exogenous provision of 5-HT. Patients with IG had a significant reduction of antral EC cell numbers and 5-HT content, which negatively correlated with gastric emptying rate. CONCLUSIONS: The Tph1CreERT2/+ mouse provides a powerful tool to study the functional roles of EC cells in the GI tract. Our findings suggest a new pathophysiologic mechanism of 5-HT deficiency in IG.

Evidence for Glucagon Secretion and Function Within the Human Gut.

Glucagon is secreted by pancreatic α cells in response to hypoglycemia and increases hepatic glucose output through hepatic glucagon receptors (GCGRs). There is evidence supporting the notion of extrapancreatic glucagon but its source and physiological functions remain elusive. Intestinal tissue samples were obtained from patients undergoing surgical resection of cancer. Mass spectrometry analysis was used to detect glucagon from mucosal lysate. Static incubations of mucosal tissue were performed to assess glucagon secretory response. Glucagon concentration was quantitated using a highly specific sandwich enzyme-linked immunosorbent assay. A cholesterol uptake assay and an isolated murine colonic motility assay were used to assess the physiological functions of intestinal GCGRs. Fully processed glucagon was detected by mass spectrometry in human intestinal mucosal lysate. High glucose evoked significant glucagon secretion from human ileal tissue independent of sodium glucose cotransporter and KATP channels, contrasting glucose-induced glucagon-like peptide 1 (GLP-1) secretion. The GLP-1 receptor agonist Exendin-4 attenuated glucose-induced glucagon secretion from the human ileum. GCGR blockade significantly increased cholesterol uptake in human ileal crypt culture and markedly slowed ex vivo colonic motility. Our findings describe the human gut as a potential source of extrapancreatic glucagon and demonstrate a novel enteric glucagon/GCGR circuit with important physiological functions beyond glycemic regulation.

Enteroendocrine cells sense bacterial tryptophan catabolites to activate enteric and vagal neuronal pathways.

The intestinal epithelium senses nutritional and microbial stimuli using epithelial sensory enteroendocrine cells (EEC). EECs communicate nutritional information to the nervous system, but whether they also relay signals from intestinal microbes remains unknown. Using in vivo real-time measurements of EEC and nervous system activity in zebrafish, we discovered that the bacteria Edwardsiella tarda activate EECs through the receptor transient receptor potential ankyrin A1 (Trpa1) and increase intestinal motility. Microbial, pharmacological, or optogenetic activation of Trpa1+EECs directly stimulates vagal sensory ganglia and activates cholinergic enteric neurons by secreting the neurotransmitter 5-hydroxytryptamine (5-HT). A subset of indole derivatives of tryptophan catabolism produced by E. tarda and other gut microbes activates zebrafish EEC Trpa1 signaling. These catabolites also directly stimulate human and mouse Trpa1 and intestinal 5-HT secretion. These results establish a molecular pathway by which EECs regulate enteric and vagal neuronal pathways in response to microbial signals.

Amperometry in Single Cells and Tissue.

The release from cells of signaling molecules through the controlled process of exocytosis involves multiple coordinated steps and is essential for the proper control of a multitude of biological pathways across the endocrine and nervous systems. However, these events are minute both temporally and in terms of the minute amounts of neurotransmitters, hormones, growth factors, and peptides released from single vesicles during exocytosis. It is therefore difficult to measure the kinetics of single exocytosis events in real time. One noninvasive method of measuring the release of molecules from cells is carbon-fiber amperometry. In this chapter, we will describe how we undertake such measurements from both single cells and in live tissue, how the subsequent data are analyzed, and how we interpret these results in terms of their relevant physiology.

Circulating cathepsin S improves glycaemic control in mice.

Cathepsin S (CTSS) is a cysteine protease that regulates many physiological processes and is increased in obesity and type 2 diabetes. While previous studies show that deletion of CTSS improves glycaemic control through suppression of hepatic glucose output, little is known about the role of circulating CTSS in regulating glucose and energy metabolism. We assessed the effects of recombinant CTSS on metabolism in cultured hepatocytes, myotubes and adipocytes, and in mice following acute CTSS administration. CTSS improved glucose tolerance in lean mice and this coincided with increased plasma insulin. CTSS reduced G6pc and Pck1 mRNA expression and glucose output from hepatocytes but did not affect glucose metabolism in myotubes or adipocytes. CTSS did not affect insulin secretion from pancreatic ß-cells, rather CTSS stimulated glucagon-like peptide (GLP)-1 secretion from intestinal mucosal tissues. CTSS retained its positive effects on glycaemic control in mice injected with the GLP1 receptor antagonist Exendin (9-39) amide. The effects of CTSS on glycaemic control were not retained in high-fat-fed mice or db/db mice, despite the preservation of CTSS' inhibitory actions on hepatic glucose output in isolated primary hepatocytes. In conclusion, we unveil a role for CTSS in the regulation of glycaemic control via direct effects on hepatocytes, and that these effects on glycaemic control are abrogated in insulin resistant states.

The Suitability of Glioblastoma Cell Lines as Models for Primary Glioblastoma Cell Metabolism.

In contrast to most non-malignant tissue, cells comprising the brain tumour glioblastoma (GBM) preferentially utilise glycolysis for metabolism via "the Warburg effect". Research into therapeutics targeting the disease's highly glycolytic state offer a promising avenue to improve patient survival. These studies often employ GBM cell lines for in vitro studies which translate poorly to the in vivo patient context. The metabolic traits of five of the most used GBM cell lines were assessed and compared to primary GBM and matched, healthy brain tissue. In patient-derived GBM cell lines, the basal mitochondrial rate (p = 0.043) and ATP-linked respiration (p < 0.001) were lower than primary adjacent normal cells from the same patient, while reserve capacity (p = 0.037) and Krebs cycle capacity (p = 0.002) were higher. Three cell lines, U251MG, U373MG and D54, replicate the mitochondrial metabolism of primary GBM cells. Surprisingly, glycolytic capacity is not different between healthy and GBM tissue. The T98G cell line recapitulated glycolysis-related metabolic parameters of the primary GBM cells and is recommended for research relating to glycolysis. These findings can guide preclinical research into the development of novel therapeutics targeting metabolic pathways in GBM.

The ever-changing roles of serotonin.

Serotonin (5-HT) has traditional roles as a key neurotransmitter in the central nervous system and as a regulatory hormone controlling a broad range of physiological functions. Perhaps the most classically-defined functions of 5-HT are centrally in the control of mood, sleep and anxiety and peripherally in the modulation of gastrointestinal motility. A more recently appreciated role for 5-HT has emerged, however, as an important metabolic hormone contributing to glucose homeostasis and adiposity, with a causal relationship existing between circulating 5-HT levels and metabolic diseases. Almost all peripheral 5-HT is derived from specialised enteroendocrine cells, called enterochromaffin (EC) cells, located throughout the length of the lining of the gastrointestinal tract. EC cells are important luminal sensory cells that can detect and respond to an array of ingested nutrients, as well as luminal gut microbiota and their associated metabolites. Intriguingly, the interaction between gut microbiota and EC cells is dynamic in nature and has strong implications for host physiology. In this review, we discuss the traditional and modern functions of 5-HT and highlight an emerging pathway by which gut microbiota influences host health. Serotonin, also known as 5-hydroxytryptamine (5-HT), is an important neurotransmitter, growth factor and hormone that mediates a range of physiological functions. In mammals, serotonin is synthesized from the essential amino acid tryptophan by the rate-limiting enzyme tryptophan hydroxylase (TPH), for which there are two isoforms expressed in distinct cell types throughout the body. Tph1 is mainly expressed by specialized gut endocrine cells known as enterochromaffin (EC) cells and by other non-neuronal cell types such as adipocytes (Walther et al., 2003). Tph2 is primarily expressed in neurons of the raphe nuclei of the brain stem and a subset of neurons in the enteric nervous system (ENS) (Yabut et al., 2019). As 5-HT cannot readily cross the blood-brain barrier, the central and peripheral pools of 5-HT are anatomically separated and as such, act in their own distinct manners (Martin et al., 2017c). In this review we discuss the peripheral roles of serotonin, with particular focus on the interaction of gut-derived serotonin with the gut microbiota, and address emerging evidence linking this relationship with host homeostasis.

Diet differentially regulates enterochromaffin cell serotonin content, density and nutrient sensitivity in the mouse small and large intestine.

BACKGROUND: Enterochromaffin (EC) cells are specialized enteroendocrine cells lining the gastrointestinal (GI) tract and the source of almost all serotonin (5-hydroxytryptamine; 5-HT) in the body. Gut-derived 5-HT has a plethora of physiological roles, including regulation of gastrointestinal motility, and has been implicated as a driver of obesity and metabolic disease. This is due to 5-HT influencing key metabolic processes, such as hepatic gluconeogenesis, adipose tissue lipolysis and hindering thermogenic capacity. Increased circulating 5-HT occurs in humans with obesity and type 2 diabetes. However, despite the known metabolic roles of gut-derived 5-HT, the mechanisms underlying the cellular-level change in EC cells under obesogenic conditions remains unknown. METHODS: We use a mouse model of diet-induced obesity (DIO) to identify the regional changes that occur in primary EC cells from the duodenum and colon. Transcriptional changes in the nutrient sensing profile of primary EC cells were assessed, and responses to nutrient stimuli in culture were determined by 5-HT ELISA. KEY RESULTS: We find that obesogenic conditions affect EC cells in a region-dependent manner. Duodenal EC cells from DIO mice have impaired sugar sensing even in the presence of increased 5-HT content per cell, while colonic EC cell numbers are significantly increased, but have unaltered nutrient sensing capacity. CONCLUSIONS & INFERENCES: Our findings from this study add novel insights into the mechanisms by which functional changes to EC cells occur at a cellular level, which may contribute to the altered circulating 5-HT seen with obesity and metabolic disease, and associated gastrointestinal disorders.