Signalling pathways involved in the detection of peptones by murine small intestinal enteroendocrine L-cells.

Glucagon like peptide-1 is an insulinotropic hormone released from intestinal L-cells in response to food ingestion. Here, we investigated mechanisms underlying the sensing of peptones by primary small intestinal L-cells. Meat, casein and vegetable-derived peptones (5 mg/ml), the L-amino acids Phe, Trp, Gln and Ala (20 mM each), and the dipeptide glycine-sarcosine (20 mM) stimulated GLP-1 secretion from primary cultures prepared from the small intestine. Further mechanistic studies were performed with meat peptone, and revealed the elevation of intracellular calcium in L-cells. Inhibition of the calcium sensing receptor (CaSR), transient receptor potential (TRP) channels and Q-type voltage gated calcium channels (VGCC) significantly attenuated peptone-stimulated GLP-1 release and reduced intracellular Ca(2+) responses. CaSR inhibition also attenuated the GLP-1 secretory response to Gln. Targeting these pathways in L-cells could be used to increase endogenous production of GLP-1 and offer exploitable avenues for the development of therapeutics to treat diabetes and obesity.This work was funded by grants from the Wellcome Trust (WT088357/Z/09/Z and WT084210/Z/07/Z), the MRC Metabolic Diseases Unit (MRC_MC_UU_12012/3) and Full4Health (FP7/2011-2015, grant agreement n° 266408).This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.peptides.2015.07.01

The role of gut endocrine cells in control of metabolism and appetite.

After food is ingested, nutrients pass through the gastrointestinal tract, stimulating the release of a range of peptide hormones. Among their many local, central and peripheral actions, these hormones act to mediate glucose metabolism and satiety. Indeed, it is the modification of gut hormone secretion that is considered partly responsible for the normalization of glycaemic control and the reduction in appetite seen in many patients after certain forms of bariatric surgery. This review describes recent developments in our understanding of the secretion and action of anorexigenic gut hormones, primarily concentrating on glucagon-like peptide-1 (GLP-1).This is the final version. It was first published by Wiley in Experimental Physiology here: http://onlinelibrary.wiley.com/doi/10.1113/expphysiol.2014.079764/abstract

Nutrient-Induced Cellular Mechanisms of Gut Hormone Secretion.

The gastrointestinal tract can assess the nutrient composition of ingested food. The nutrient-sensing mechanisms in specialised epithelial cells lining the gastrointestinal tract, the enteroendocrine cells, trigger the release of gut hormones that provide important local and central feedback signals to regulate nutrient utilisation and feeding behaviour. The evidence for nutrient-stimulated secretion of two of the most studied gut hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), along with the known cellular mechanisms in enteroendocrine cells recruited by nutrients, will be the focus of this review. The mechanisms involved range from electrogenic transporters, ion channel modulation and nutrient-activated G-protein coupled receptors that converge on the release machinery controlling hormone secretion. Elucidation of these mechanisms will provide much needed insight into postprandial physiology and identify tractable dietary approaches to potentially manage nutrition and satiety by altering the secreted gut hormone profile

Models and Tools for Studying Enteroendocrine Cells.

Gut hormones produced by gastrointestinal enteroendocrine cells modulate key physiological processes including glucose homeostasis and food intake, making them potential therapeutic candidates to treat obesity and diabetes. Understanding the function of enteroendocrine cells and the molecular mechanisms driving hormone production is a key step toward mobilizing endogenous hormone reserves in the gut as a therapeutic strategy. In this review, we will discuss the variety of ex vivo and in vitro model systems driving this research and their contributions to our current understanding of nutrient-sensing mechanisms in enteroendocrine cells

Mechanisms underlying glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 secretion.

The incretin hormones, glucose-dependent insulinotropic peptide and glucagon-like peptide-1, are secreted from intestinal K- and L cells, respectively, with the former being most abundant in the proximal small intestine, whereas the latter increase in number towards the distal gut. Although an overlap between K- and L cells can be observed immunohistochemically or in murine models expressing fluorescent markers under the control of the two hormone promoters, the majority (>80%) of labeled cells seems to produce only one of these hormones. Transcriptomic analysis showed a close relationship between small intestinal K- and L cells, and glucose sensing mechanisms appear similar in both cell types with a predominant role of electrogenic glucose uptake through sodium-coupled glucose transporter 1. Similarly, both cell types produce the long-chain fatty acid sensing G-protein-coupled receptors, FFAR1 (GPR40) and FFAR4 (GPR120), but differ in the expression/functionality of other lipid sensing receptors. GPR119 and FFAR2/3, for example, have clearly documented roles in glucagon-like peptide-1 secretion, whereas agonists for the endocannabinoid receptor type 1 have been found to show largely selective inhibition of glucose-dependent insulinotropic peptide secretion. In conclusion, although K- and L cell populations overlap and share key molecular nutrient-sensing mechanisms, subtle differences between the responsiveness of the different cell types might be exploited to differentially modulate glucose-dependent insulinotropic peptide or glucagon-like peptide-1 secretion

Bacterial metabolite indole modulates incretin secretion from intestinal enteroendocrine L cells.

It has long been speculated that metabolites, produced by gut microbiota, influence host metabolism in health and diseases. Here, we reveal that indole, a metabolite produced from the dissimilation of tryptophan, is able to modulate the secretion of glucagon-like peptide-1 (GLP-1) from immortalized and primary mouse colonic L cells. Indole increased GLP-1 release during short exposures, but it reduced secretion over longer periods. These effects were attributed to the ability of indole to affect two key molecular mechanisms in L cells. On the one hand, indole inhibited voltage-gated K(+) channels, increased the temporal width of action potentials fired by L cells, and led to enhanced Ca(2+) entry, thereby acutely stimulating GLP-1 secretion. On the other hand, indole slowed ATP production by blocking NADH dehydrogenase, thus leading to a prolonged reduction of GLP-1 secretion. Our results identify indole as a signaling molecule by which gut microbiota communicate with L cells and influence host metabolism.This is the final version. It was first published by Elsevier at http://www.cell.com/cell-reports/abstract/S2211-1247%2814%2900901-2

Mass spectrometric characterisation of the circulating peptidome following oral glucose ingestion in control and gastrectomised patients.

RATIONALE: Meal ingestion triggers secretion of a variety of gut and endocrine peptides important in diabetes research which are routinely measured by immunoassays. However, similarities between some peptides (glucagon, oxyntomodulin and glicentin) can cause specificity issues with immunoassays. We used a liquid chromatography/tandem mass spectrometry (LC/MS/MS) methodology to unambiguously monitor multiple gut peptides in human plasma. METHODS: A simple acetonitrile-based protein precipitation step, followed by evaporation and solid-phase extraction, removed high-abundance proteins from samples prior to nano-LC/MS/MS analysis on an Orbitrap Q-Exactive Plus mass spectrometer using a data-dependent methodology. Database searching using PEAKS identified multiple gut-derived peptides, including peptides in the mid-pg/mL range. The relative levels of these and previously characterised peptides were assessed in plasma samples from gastrectomised and control subjects during an oral glucose tolerance test. RESULTS: Analysis of plasma extracts revealed significantly elevated levels of a number of peptides following glucose ingestion in subjects who had undergone gastrectomy compared with controls. These included GLP-1(7-36), GLP-1(9-36), glicentin, oxyntomodulin, GIP(1-42), GIP(3-42), PYY(1-36), PYY(3-36), neurotensin, insulin and C-peptide. Motilin levels decreased following glucose ingestion. Results showed good correlation with immunoassay-derived concentrations of some peptides in the same samples. The gastrectomy group also had higher, but non-glucose-dependent, circulating levels of peptides from PIGR and DMBT1. CONCLUSIONS: Overall, the approach showed that a fast, generic and reproducible LC/MS/MS methodology requiring only a small volume of plasma was capable of the multiplexed detection of a variety of diabetes-related peptides.Wellcome Trust and MR

Novel SCN9A mutations underlying extreme pain phenotypes: unexpected electrophysiological and clinical phenotype correlations.

The importance of NaV1.7 (encoded by SCN9A) in the regulation of pain sensing is exemplified by the heterogeneity of clinical phenotypes associated with its mutation. Gain-of-function mutations are typically pain-causing and have been associated with inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD). IEM is usually caused by enhanced NaV1.7 channel activation, whereas mutations that alter steady-state fast inactivation often lead to PEPD. In contrast, nonfunctional mutations in SCN9A are known to underlie congenital insensitivity to pain (CIP). Although well documented, the correlation between SCN9A genotypes and clinical phenotypes is still unclear. Here we report three families with novel SCN9A mutations. In a multiaffected dominant family with IEM, we found the heterozygous change L245 V. Electrophysiological characterization showed that this mutation did not affect channel activation but instead resulted in incomplete fast inactivation and a small hyperpolarizing shift in steady-state slow inactivation, characteristics more commonly associated with PEPD. In two compound heterozygous CIP patients, we found mutations that still retained functionality of the channels, with two C-terminal mutations (W1775R and L1831X) exhibiting a depolarizing shift in channel activation. Two mutations (A1236E and L1831X) resulted in a hyperpolarizing shift in steady-state fast inactivation. To our knowledge, these are the first descriptions of mutations with some retained channel function causing CIP. This study emphasizes the complex genotype-phenotype correlations that exist for SCN9A and highlights the C-terminal cytoplasmic region of NaV1.7 as a critical region for channel function, potentially facilitating analgesic drug development studies.J.J.C. and A.M.H. were supported by an MRC Research Career Development fellowship. F.M.G., F.R., and E.C.E. were supported by Wellcome Trust Senior Fellowships WT088357/Z/09/Z and WT084210/Z/07/Z and MRC Grant MC_UU_12012/3. C.G.W. was supported by the Cambridge Biomedical Research Campus.This is the final published version. It first appeared at http://www.jneurosci.org/content/35/20/7674.short