Sugar responses of human enterochromaffin cells depend on gut region, sex, and body mass

Gut-derived serotonin (5-HT) is released from enterochromaffin (EC) cells in response to nutrient cues, and acts to slow gastric emptying and modulate gastric motility. Rodent studies also evidence a role for gut-derived 5-HT in the control of hepatic glucose production, lipolysis and thermogenesis, and in mediating diet-induced obesity. EC cell number and 5-HT content is increased in the small intestine of obese rodents and human, however, it is unknown whether EC cells respond directly to glucose in humans, and whether their capacity to release 5-HT is perturbed in obesity. We therefore investigated 5-HT release from human duodenal and colonic EC cells in response to glucose, sucrose, fructose and α-glucoside (αMG) in relation to body mass index (BMI). EC cells released 5-HT only in response to 100 and 300 mM glucose (duodenum) and 300 mM glucose (colon), independently of osmolarity. Duodenal, but not colonic, EC cells also released 5-HT in response to sucrose and αMG, but did not respond to fructose. 5-HT content was similar in all EC cells in males, and colonic EC cells in females, but 3 to 4-fold higher in duodenal EC cells from overweight females (p < 0.05 compared to lean, obese). Glucose-evoked 5-HT release was 3-fold higher in the duodenum of overweight females (p < 0.05, compared to obese), but absent here in overweight males. Our data demonstrate that primary human EC cells respond directly to dietary glucose cues, with regional differences in selectivity for other sugars. Augmented glucose-evoked 5-HT release from duodenal EC is a feature of overweight females, and may be an early determinant of obesity.Amanda L. Lumsden, Alyce M. Martin, Emily W. Sun, Gudrun Schober, Nicole J. Isaacs, Nektaria Pezos, David A. Wattchow, Dayan de Fontgalland, Philippa Rabbitt, Paul Hollington, Luigi Sposato, Steven L. Due, Christopher K. Rayner, Nam Q. Nguyen, Alice P. Liou, V. Margaret Jackson, Richard L. Young, and Damien J. Keatin

The neurochemical changes in the innervation of human colonic mesenteric and submucosal blood vessels in Ulcerative colitis and Crohn's disease

BACKGROUND: Neurogenic inflammation involves vasodilation, oedema and sensory nerve hypersensitivity. Extrinsic sensory nerves to the intestinal wall mediate these effects and functional subsets of these extrinsic nerves can be characterized by immunohistochemical profiles. In this study such profiles were examined in samples from patients with inflammatory bowel disease (IBD), in particular ulcerative colitis (UC) and Crohn's disease (CD). METHODS: Healthy margins from cancer patients were compared to specimens from IBD patients. All nerve fibres were labelled by PGP 9.5. Double and triple labelling with TH, NPY, SP, SOM, NOS, VIP, VAChT, CGRP, TRPv1 were performed. Perivascular nerve fibres in the mesentery, and submucosa, were examined. The percentage of all labelled nerve fibres was calculated with a transect method. KEY RESULTS: Total number of varicosities on mesenteric vessels increased in IBD but decreased around submucosal vessels. The percentage of nerve fibres around submucosal arteries labelled by SP increased from 11% in controls to 20% (UC) and 24% (CD) and mesenteric artery nerve fibres were unchanged. Nerve fibres labelled by SOM were markedly reduced surrounding submucosal arteries, from 22% to 1% (UC) and 2% (CD), but not perivascular mesenteric nerve fibres. 87 to 93% of SP immunoreactive nerve fibres were also reactive for TRvP1. TRPv1 labelling without SP was 12%in controls and increased to 40% in CD submucosal specimens. CONCLUSIONS & INFERENCES: There is an increase in SP and TRPv1, and a reduction in SOM immunoreactive nerve fibres in IBD. Changes in the perivascular functional nerve subclasses may underlie the hyperaemia, and ulceration, characteristic of IBD. Furthermore, pain may relate to underlying neural changes.D. de Fontgalland, S. J. Brookes, I. Gibbins, T. C. Sia and D. A. Wattcho

Mechanisms controlling glucose-induced GLP-1 secretion in human small intestine

Intestinal glucose stimulates secretion of the incretin hormone glucagon-like peptide 1 (GLP-1). The mechanisms underlying this pathway have not been fully investigated in humans. In this study, we showed that a 30-min intraduodenal glucose infusion activated half of all duodenal L cells in humans. This infusion was sufficient to increase plasma GLP-1 levels. With an ex vivo model using human gut tissue specimens, we showed a dose-responsive GLP-1 secretion in the ileum at ≥200 mmol/L glucose. In ex vivo tissue from the duodenum and ileum, but not the colon, 300 mmol/L glucose potently stimulated GLP-1 release. In the ileum, this response was independent of osmotic influences and required delivery of glucose via GLUT2 and mitochondrial metabolism. The requirement of voltage-gated Na⁺ and Ca²⁺ channel activation indicates that membrane depolarization occurs. KATP channels do not drive this, as tolbutamide did not trigger release. The sodium-glucose cotransporter 1 (SGLT1) substrate α-MG induced secretion, and the response was blocked by the SGLT1 inhibitor phlorizin or by replacement of extracellular Na⁺ with N-methyl-d-glucamine. This is the first report of the mechanisms underlying glucose-induced GLP-1 secretion from human small intestine. Our findings demonstrate a dominant role of SGLT1 in controlling glucose-stimulated GLP-1 release in human ileal L cells.Emily W. Sun, Dayan de Fontgalland, Philippa Rabbitt, Paul Hollington, Luigi Sposato, Steven L. Due, David A. Wattchow, Christopher K. Rayner, Adam M. Deane, Richard L. Young, and Damien J. Keatin

Evidence for glucagon secretion and function within the human gut

Glucagon is secreted by pancreatic α cells in response to hypoglycaemia and increases hepatic glucose output through hepatic glucagon receptors (GCGR). There is evidence supporting the notion of extra-pancreatic glucagon but its source and physiological functions remain elusive. Intestinal tissue 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 ELISA. A cholesterol uptake assay and an isolated murine colonic motility assay were used to assess the physiological functions of intestinal GCGR. 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 SGLT 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 glycaemic regulation.Emily W Sun, Alyce M Martin, Dayan de Fontgalland, Luigi Sposato, Philippa Rabbitt, Paul Hollington ... et al

A gut-intrinsic melanocortin signalling 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 GLP-1 and 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 BMI-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 (POMC) 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 signalling nexus in 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.Emily W. Sun, Eva W. Iepsen, Nektaria Pezos, Amanda L. Lumsden, Alyce M. Martin, Gudrun Schober, Nichole J. Isaacs, Christopher K. Rayner, Nam Q. Nguyen, Dayan de Fontgalland, Philippa Rabbitt, Paul Hollington, David A. Wattchow, Torben Hansen, Jens-Christian Holm, Alice P. Liou, V. Margaret Jackson, Signe S. Torekov, Richard L. Young, and Damien J. Keatin

Metformin-induced glucagon-like peptide-1 secretion contributes to the actions of metformin in type 2 diabetes

Background: Metformin reduces plasma glucose and has been shown to increase glucagon-like peptide 1 (GLP-1) secretion. Whether this is a direct action of metformin on GLP-1 release, and whether some of the glucose-lowering effect of metformin occurs due to GLP-1 release, is unknown. The current study investigated metformin-induced GLP-1 secretion and its contribution to the overall glucose-lowering effect of metformin and underlying mechanisms in patients with type 2 diabetes. Methods: Twelve patients with type 2 diabetes were included in this placebo-controlled, double-blinded study. On 4 separate days, the patients received metformin (1,500 mg) or placebo suspended in a liquid meal, with subsequent i.v. infusion of the GLP-1 receptor antagonist exendin9-39 (Ex9-39) or saline. During 240 minutes, blood was sampled. The direct effect of metformin on GLP-1 secretion was tested ex vivo in human ileal and colonic tissue with and without dorsomorphin-induced inhibiting of the AMPK activity. Results: Metformin increased postprandial GLP-1 secretion compared with placebo (P = 0.014), and the postprandial glucose excursions were significantly smaller after metformin + saline compared with metformin + Ex9-39 (P = 0.004). Ex vivo metformin acutely increased GLP-1 secretion (colonic tissue, P < 0.01; ileal tissue, P < 0.05), but the effect was abolished by inhibition of AMPK activity. Conclusions: Metformin has a direct and AMPK-dependent effect on GLP-1-secreting L cells and increases postprandial GLP-1 secretion, which seems to contribute to metformin's glucose-lowering effect and mode of action.Emilie Bahne, Emily W.L. Sun, Richard L. Young, Morten Hansen, David P. Sonne, Jakob S. Hansen, Ulrich Rohde, Alice P. Liou, Margaret L. Jackson, Dayan de Fontgalland, Philippa Rabbitt, Paul Hollington, Luigi Sposato, Steven Due, David A. Wattchow, Jens F. Rehfeld, Jens J. Holst, Damien J. Keating, Tina Vilsbøll, and Filip K. Kno

Immunoreactivity for the high-affinity choline transporter colocalises with VAChT in the human enteric nervous system

Cholinergic nerves are identified by labelling molecules in the ACh synthesis, release and destruction pathway. Recently, antibodies against another molecule in this pathway have been developed. Choline reuptake at the synapse occurs via the high-affinity choline transporter (CHT1). CHT1 immunoreactivity is present in cholinergic nerve fibres containing vesicular acetylcholine transporter (VAChT) in the human and rat central nervous system and rat enteric nervous system. We have examined whether CHT1 immunoreactivity is present in nerve fibres in human intestine and whether it is colocalised with markers of cholinergic, tachykinergic or nitrergic circuitry. Human ileum and colon were fixed, sectioned and processed for fluorescence immunohistochemistry with antibodies against CHT1, class III beta-tubulin (TUJ1), synaptophysin, common choline acetyl-transferase (cChAT), VAChT, nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). CHT1 immunoreactivity was present in many nerve fibres in the circular and longitudinal muscle, myenteric and submucosal ganglia, submucosa and mucosa in human colon and ileum and colocalised with immunoreactivity for TUJ1 and synaptophysin confirming its presence in nerve fibres. In nerve fibres in myenteric ganglia and muscle, CHT1 immunoreactivity colocalised with immunoreactivity for VAChT and cChAT. Some colocalisation occurred with SP immunoreactivity, but little with immunoreactivity for VIP or NOS. In the mucosa, CHT1 immunoreactivity colocalised with that for VIP and SP in nerve fibres and was also present in vascular nerve fibres in the submucosa and on epithelial cells on the luminal border of crypts. The colocalisation of CHT1 immunoreactivity with VAChT immunoreactivity in cholinergic enteric nerves in the human bowel thus suggests that CHT1 represents another marker of cholinergic nerves.Andrea M. Harrington, Margaret Lee, Sim-Yee Ong, Eric Yong, Pamela Farmer, Cristal J. Peck, Chung W. Chow, John M. Hutson and Bridget R. Southwel