Oligopeptides stimulate glucagon-like peptide-1 secretion in mice through proton-coupled uptake and the calcium-sensing receptor.

AIMS/HYPOTHESIS: Ingested protein is a well-recognised stimulus for glucagon-like peptide-1 (GLP-1) release from intestinal L cells. This study aimed to characterise the molecular mechanisms employed by L cells to detect oligopeptides. METHODS: GLP-1 secretion from murine primary colonic cultures and Ca(2+) dynamics in L cells were monitored in response to peptones and dipeptides. L cells were identified and purified based on their cell-specific expression of the fluorescent protein Venus, using GLU-Venus transgenic mice. Pharmacological tools and knockout mice were used to characterise candidate sensory pathways identified by expression analysis. RESULTS: GLP-1 secretion was triggered by peptones and di-/tripeptides, including the non-metabolisable glycine-sarcosine (Gly-Sar). Two sensory mechanisms involving peptide transporter-1 (PEPT1) and the calcium-sensing receptor (CaSR) were distinguishable. Responses to Gly-Sar (10 mmol/l) were abolished in the absence of extracellular Ca(2+) or by the L-type calcium-channel blocker nifedipine (10 µmol/l) and were PEPT1-dependent, as demonstrated by their sensitivity to pH and 4-aminomethylbenzoic acid and the finding of impaired responses in tissue from Pept1 (also known as Slc15a1) knockout mice. Peptone (5 mg/ml)-stimulated Ca(2+) responses were insensitive to nifedipine but were blocked by antagonists of CaSR. Peptone-stimulated GLP-1 secretion was not impaired in mice lacking the putative peptide-responsive receptor lysophosphatidic acid receptor 5 (LPAR5; also known as GPR92/93). CONCLUSIONS/INTERPRETATION: Oligopeptides stimulate GLP-1 secretion through PEPT1-dependent electrogenic uptake and activation of CaSR. Both pathways are highly expressed in native L cells, and likely contribute to the ability of ingested protein to elevate plasma GLP-1 levels. Targeting nutrient-sensing pathways in L cells could be used to mobilise endogenous GLP-1 stores in humans, and could mimic some of the metabolic benefits of bariatric surgery.

Intestinal sensing of nutrients.

Ingestion of a meal triggers a range of physiological responses both within and outside the gut, and results in the remote modulation of appetite and glucose homeostasis. Luminal contents are sensed by specialised chemosensitive cells scattered throughout the intestinal epithelium. These enteroendocrine and tuft cells make direct contact with the gut lumen and release a range of chemical mediators, which can either act in a paracrine fashion interacting with neighbouring cells and nerve endings or as classical circulating hormones. At the molecular level, the chemosensory machinery involves multiple and complex signalling pathways including activation of G-protein-coupled receptors and solute carrier transporters. This chapter will discuss our current knowledge of the molecular mechanisms underlying intestinal chemosensation with a particular focus on the relatively well-characterised nutrient-triggered secretion from the enteroendocrine system.

Nutritional regulation of glucagon-like peptide-1 secretion.

Glucagon-like peptide-1 (GLP-1), released from L-cells in the intestinal epithelium, plays an important role in postprandial glucose homeostasis and appetite control. Following the recent therapeutic successes of antidiabetic drugs aimed at either mimicking GLP-1 or preventing its degradation, attention is now turning towards the L-cell, and addressing whether it would be both possible and beneficial to stimulate the endogenous release of GLP-1 in vivo. Understanding the mechanisms underlying GLP-1 release from L-cells is key to this type of approach, and the use of cell line models has led to the identification of a variety of pathways that may underlie the physiological responses of L-cells to food ingestion. This review focuses on our current understanding of the signalling mechanisms that underlie L-cell nutrient responsiveness.

Adenosine triphosphate is co-secreted with glucagon-like peptide-1 to modulate intestinal enterocytes and afferent neurons.

Enteroendocrine cells are specialised sensory cells located in the intestinal epithelium and generate signals in response to food ingestion. Whilst traditionally considered hormone-producing cells, there is evidence that they also initiate activity in the afferent vagus nerve and thereby signal directly to the brainstem. We investigate whether enteroendocrine L-cells, well known for their production of the incretin hormone glucagon-like peptide-1 (GLP-1), also release other neuro-transmitters/modulators. We demonstrate regulated ATP release by ATP measurements in cell supernatants and by using sniffer patches that generate electrical currents upon ATP exposure. Employing purinergic receptor antagonists, we demonstrate that evoked ATP release from L-cells triggers electrical responses in neighbouring enterocytes through P2Y2 and nodose ganglion neurones in co-cultures through P2X2/3-receptors. We conclude that L-cells co-secrete ATP together with GLP-1 and PYY, and that ATP acts as an additional signal triggering vagal activation and potentially synergising with the actions of locally elevated peptide hormone concentrations.

Expression profiling and electrophysiological studies suggest a major role for Orai1 in the store-operated Ca2+ influx pathway of platelets and megakaryocytes.

Store-operated Ca2+ influx represents a major route by which cytosolic Ca2+ can be elevated during platelet activation, yet its molecular identity in this cell type remains highly controversial. Using quantitative RT-PCR analysis of candidate receptor-operated cation entry pathways in human platelets, we show a >30-fold higher expression of message for the recently discovered Orai1 store-operated Ca2+ channel, and also the store Ca2+ sensor STIM1, when compared to the non-selective cation channels TRPC1, TRPC6 and TRPM2. Orai1 and STIM1 gene transcripts were also detected at higher levels than TRPC1, TRPC6 and TRPM2 in primary murine megakaryocytes and human megakaryocytic cell lines. In direct electrophysiological recordings from murine megakaryocytes, Ca2+ ionophore-induced store depletion stimulated CRAC currents, which are known to require Orai1, and these overlapped with TRPC6-like currents following P2Y receptor activation. Together with recent transgenic studies, these data provide evidence for STIM1:Orai1 as a primary pathway for agonist-evoked Ca2+ influx in the platelet and megakaryocyte.

Mechanistic insights into the detection of free fatty and bile acids by ileal glucagon-like peptide-1 secreting cells.

OBJECTIVES: The aim of this study was to investigate the electrical properties of ileal Glucagon-like peptide 1 (GLP-1) secreting L-cells using murine organoid cultures and the electrophysiological and intracellular signaling pathways recruited following activation of the Gαq-coupled free fatty acid receptors FFA1 and Gαs-coupled bile acid receptors GPBAR1. METHODS: Experiments were performed using ileal organoids generated from mice transgenically expressing fluorescent reporters (Epac2-camps and GCaMP3) under control of the proglucagon promoter. Electrophysiology and single cell imaging were performed on identified L-cells in organoids, and GLP-1 secretion from cultured organoids was measured by immunoassay. RESULTS: The FFA1 ligand TAK-875 triggered L-cell electrical activity, increased intracellular calcium, and activated a depolarizing current that was blocked by the TRPC3 inhibitor Pyr3. TAK-875 triggered GLP-1 secretion was Pyr3 sensitive, suggesting that the TRPC3 channel links FFA1 activation to calcium elevation and GLP-1 release in L-cells. GPBAR1 agonist triggered PKA-dependent L-type Ca2+ current activation and action potential firing in L-cells. The combination of TAK-875 and a GPBAR1 agonist triggered synergistic calcium elevation and GLP-1 secretory responses. CONCLUSIONS: FFA1 and GPBAR1 activation individually increased electrical activity in L-cells by recruiting pathways that include activation of TRPC3 and L-type voltage-gated Ca2+ channels. Synergy between the pathways activated downstream of these receptors was observed both at the level of Ca2+ elevation and GLP-1 secretion.

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells.

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

G-protein-coupled receptors in intestinal chemosensation.

Food intake is detected by the chemical senses of taste and smell and subsequently by chemosensory cells in the gastrointestinal tract that link the composition of ingested foods to feedback circuits controlling gut motility/secretion, appetite, and peripheral nutrient disposal. G-protein-coupled receptors responsive to a range of nutrients and other food components have been identified, and many are localized to intestinal chemosensory cells, eliciting hormonal and neuronal signaling to the brain and periphery. This review examines the role of G-protein-coupled receptors as signaling molecules in the gut, with a particular focus on pathways relevant to appetite and glucose homeostasis.

Purification of native bone marrow megakaryocytes for studies of gene expression.

Megakaryocytes constitute less than 1% of all marrow cells, therefore purification of these giant platelet precursor cells represents a challenge. We describe two methods to ultra-purify mature megakaryocytes from murine marrow for the purpose of extracting RNA suitable for studies of gene expression. In the first approach, unit velocity gradients are used to enrich for megakaryocytes, which are then selected by fluorescence-activated cell sorting based upon size and high surface expression of CD41. In the second method, individual megakaryocytes, identified by their distinct morphology, are extracted using glass suction pipettes. Despite the small numbers of cells that can be isolated via the latter technique, recent studies have demonstrated how this pure population can be used to detect mRNA transcripts encoding ion channels and other proteins in the native megakaryocyte.

Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2.

Interest in how the gut microbiome can influence the metabolic state of the host has recently heightened. One postulated link is bacterial fermentation of "indigestible" prebiotics to short-chain fatty acids (SCFAs), which in turn modulate the release of gut hormones controlling insulin release and appetite. We show here that SCFAs trigger secretion of the incretin hormone glucagon-like peptide (GLP)-1 from mixed colonic cultures in vitro. Quantitative PCR revealed enriched expression of the SCFA receptors ffar2 (grp43) and ffar3 (gpr41) in GLP-1-secreting L cells, and consistent with the reported coupling of GPR43 to Gq signaling pathways, SCFAs raised cytosolic Ca2+ in L cells in primary culture. Mice lacking ffar2 or ffar3 exhibited reduced SCFA-triggered GLP-1 secretion in vitro and in vivo and a parallel impairment of glucose tolerance. These results highlight SCFAs and their receptors as potential targets for the treatment of diabetes.

Glutamine triggers and potentiates glucagon-like peptide-1 secretion by raising cytosolic Ca2+ and cAMP.

L-glutamine stimulates glucagon-like peptide 1 (GLP-1) secretion in human subjects and cell lines. As recent advances have enabled the study of primary GLP-1-releasing L cells, this study aimed to characterize glutamine-sensing pathways in native murine L cells. L cells were identified using transgenic mice with cell-specific expression of fluorescent markers. Cells were studied in primary colonic cultures from adult mice, or purified by flow cytometry for expression analysis. Intracellular Ca(2+) was monitored in cultures loaded with Fura2, and cAMP was studied using Förster resonance energy transfer sensors expressed in GLUTag cells. Asparagine, phenylalanine, and glutamine (10 mm) triggered GLP-1 release from primary cultures, but glutamine was the most efficacious, increasing secretion 1.9-fold with an EC(50) of 0.19 mm. Several amino acids triggered Ca(2+) changes in L cells, comparable in magnitude to that induced by glutamine. Glutamine-induced Ca(2+) responses were abolished in low Na(+) solution and attenuated in Ca(2+) free solution, suggesting a role for Na(+) dependent uptake and Ca(2+) influx. The greater effectiveness of glutamine as a secretagogue was paralleled by its ability to increase cAMP in GLUTag cells. Glutamine elevated intracellular cAMP to 36% of that produced by a maximal stimulus, whereas asparagine only increased intracellular cAMP by 24% and phenylalanine was without effect. Glutamine elevates both cytosolic Ca(2+) and cAMP in L cells, which may account for the effectiveness of glutamine as a GLP-1 secretagogue. Therapeutic agents like glutamine that target synergistic pathways in L cells might play a future role in the treatment of type 2 diabetes.

Role of phosphodiesterase and adenylate cyclase isozymes in murine colonic glucagon-like peptide 1 secreting cells.

BACKGROUND AND PURPOSE: Glucagon-like peptide-1 (GLP-1) is secreted from enteroendocrine L-cells after food intake. Increasing GLP-1 signalling either through inhibition of the GLP-1 degrading enzyme dipeptidyl-peptidase IV or injection of GLP-1-mimetics has recently been successfully introduced for the treatment of type 2 diabetes. Boosting secretion from the L-cell has so far not been exploited, due to our incomplete understanding of L-cell physiology. Elevation of cyclic adenosine monophosphate (cAMP) has been shown to be a strong stimulus for GLP-1 secretion and here we investigate the activities of adenylate cyclase (AC) and phosphodiesterase (PDE) isozymes likely to shape cAMP responses in L-cells. EXPERIMENTAL APPROACH: Expression of AC and PDE isoforms was quantified by RT-PCR. Single cell responses to stimulation or inhibition of AC and PDE isoforms were monitored with real-time cAMP probes. GLP-1 secretion was assessed by elisa. KEY RESULTS: Quantitative PCR identified expression of protein kinase C- and Ca²+-activated ACs, corresponding with phorbolester and cytosolic Ca²+-stimulated cAMP elevation. Inhibition of PDE2, 3 and 4 were found to stimulate GLP-1 secretion from murine L-cells in primary culture. This corresponded with cAMP elevations monitored with a plasma membrane targeted cAMP probe. Inhibition of PDE3 but not PDE2 was further shown to prevent GLP-1 secretion in response to guanylin, a peptide secreted into the gut lumen, which had not previously been implicated in L-cell secretion. CONCLUSIONS AND IMPLICATIONS: Our results reveal several mechanisms shaping cAMP responses in GLP-1 secreting cells, with some of the molecular components specifically expressed in L-cells when compared with their epithelial neighbours, thus opening new strategies for targeting these cells therapeutically.

Hypogonadotropic hypogonadism due to a novel missense mutation in the first extracellular loop of the neurokinin B receptor.

CONTEXT: The neurokinin B (NKB) receptor, encoded by TACR3, is widely expressed within the central nervous system, including hypothalamic nuclei involved in regulating GnRH release. We have recently reported two mutations in transmembrane segments of the receptor and a missense mutation in NKB in patients with normosmic isolated hypogonadotropic hypogonadism (nIHH). PATIENTS AND METHODS: We sequenced the TACR3 gene in a family in which three siblings had nIHH. The novel mutant receptor thus identified was studied in a heterologous expression system using calcium flux as the functional readout. RESULTS: All affected siblings were homozygous for the His148Leu mutation, in the first extracellular loop of the NKB receptor. The His148Leu mutant receptor exhibited profoundly impaired signaling in response to NKB (EC(50) = 3 +/- 0.1 nm and >5 microm for wild-type and His148Leu, respectively). The location of the mutation in an extracellular part of the receptor led us also to test whether senktide, a synthetic NKB analog, may retain ability to stimulate the mutant receptor. However, the signaling activity of the His148Leu receptor in response to senktide was also severely impaired (EC(50) = 1 +/- 1 nm for wild-type and no significant response of His148Leu to 10 microm). CONCLUSIONS: Homozygosity for the TACR3 His148Leu mutation leads to failure of sexual maturation in humans, whereas signaling by the mutant receptor in vitro in response to either NKB or senktide is severely impaired. These observations further strengthen the link between NKB, the NKB receptor, and regulation of human reproductive function.

Glucose sensing in L cells: a primary cell study.

Glucagon-like peptide-1 (GLP-1) is an enteric hormone that stimulates insulin secretion and improves glycaemia in type 2 diabetes. Although GLP-1-based treatments are clinically available, alternative strategies to increase endogenous GLP-1 release from L cells are hampered by our limited physiological understanding of this cell type. By generating transgenic mice with L cell-specific expression of a fluorescent protein, we studied the characteristics of primary L cells by electrophysiology, fluorescence calcium imaging, and expression analysis and show that single L cells are electrically excitable and glucose responsive. Sensitivity to tolbutamide and low-millimolar concentrations of glucose and alpha-methylglucopyranoside, assessed in single L cells and by hormone secretion from primary cultures, suggested that GLP-1 release is regulated by the activity of sodium glucose cotransporter 1 and ATP-sensitive K(+) channels, consistent with their high expression levels in purified L cells by quantitative RT-PCR. These and other pathways identified using this approach will provide exciting opportunities for future physiological and therapeutic exploration.

Molecular and electrophysiological characterization of transient receptor potential ion channels in the primary murine megakaryocyte.

The molecular identity of platelet Ca(2+) entry pathways is controversial. Furthermore, the extent to which Ca(2+)-permeable ion channels are functional in these tiny, anucleate cells is difficult to assess by direct electrophysiological measurements. Recent work has highlighted how the primary megakaryocyte represents a bona fide surrogate for studies of platelet signalling, including patch clamp recordings of ionic conductances. We have now screened for all known members of the transient receptor potential (TRP) family of non-selective cation channels in murine megakaryocytes following individual selection of these rare marrow cells using glass micropipettes. RT-PCR detected messages for TRPC6 and TRPC1, which have been reported in platelets and megakaryocytic cell lines, and TRPM1, TRPM2 and TRPM7, which to date have not been demonstrated in cells of megakaryocytic/platelet lineage. Electrophysiological recordings demonstrated the presence of functional TRPM7, a constitutively active cation channel sensitive to intracellular Mg(2+), and TRPM2, an ADP-ribose-dependent cation channel activated by oxidative stress. In addition, the electrophysiological and pharmacological properties of the non-selective cation channels stimulated by the physiological agonist ADP are consistent with a major role for TRPC6 in this G-protein-coupled receptor-dependent Ca(2+) influx pathway. This study defines for the first time the principal TRP channels within the primary megakaryocyte, which represent candidates for Ca(2+) influx pathways activated by a diverse range of stimuli in the platelet and megakaryocyte.

Interplay between P2Y(1), P2Y(12), and P2X(1) receptors in the activation of megakaryocyte cation influx currents by ADP: evidence that the primary megakaryocyte represents a fully functional model of platelet P2 receptor signaling.

The difficulty of conducting electrophysiologic recordings from the platelet has restricted investigations into the role of ion channels in thrombosis and hemostasis. We now demonstrate that the well-established synergy between P2Y(1) and P2Y(12) receptors during adenosine diphosphate (ADP)-dependent activation of the platelet alpha(IIb)beta(3) integrin also exists in murine marrow megakaryocytes, further supporting the progenitor cell as a bona fide model of platelet P2 receptor signaling. In patch clamp recordings, ADP (30 microM) stimulated a transient inward current at -70 mV, which was carried by Na(+) and Ca(2+) and was amplified by phenylarsine oxide, a potentiator of certain transient receptor potential (TRP) ion channels by phosphatidylinositol 4,5-bisphosphate depletion. This initial current decayed to a sustained phase, upon which repetitive transient inward cation currents with pre-dominantly P2X(1)-like kinetics were super-imposed. Abolishing P2X(1)-receptor activity prevented most of the repetitive currents, consistent with their activation by secreted adenosine triphosphate (ATP). Recordings in P2Y(1)-receptor-deficient megakaryocytes demonstrated an essential requirement of this receptor for activation of all ADP-evoked inward currents. However, P2Y(12) receptors, through the activation of PI3-kinase, played a synergistic role in both P2Y(1) and P2X(1)-receptor-dependent currents. Thus, direct stimulation of P2Y(1) and P2Y(12) receptors, together with autocrine P2X(1) activation, is responsible for the activation of nonselective cation currents by the platelet agonist ADP.

Emerging roles for P2X1 receptors in platelet activation.

The platelet surface membrane possesses three P2 receptors activated by extracellular adenosine nucleotides; one member of the ionotropic receptor family (P2X(1)) and two members of the G-protein-coupled receptor family (P2Y(1) and P2Y(12)). P2Y(1) and P2Y(12) receptors have firmly established roles in platelet activation during thrombosis and haemostasis, whereas the importance of the P2X(1) receptor has been more controversial. However, recent studies have demonstrated that P2X(1) receptors can generate significant functional platelet responses alone and in synergy with other receptor pathways. In addition, studies in transgenic animals indicate an important role for P2X(1) receptors in platelet activation, particularly under conditions of shear stress and thus during arterial thrombosis. This review discusses the background behind discovery of P2X(1) receptors in platelets and their precursor cell, the megakaryocyte, and how signalling via these ion channels may participate in platelet activation.

Sensitivity limits for voltage control of P2Y receptor-evoked Ca2+ mobilization in the rat megakaryocyte.

G-protein-coupled receptor signalling has been suggested to be voltage dependent in a number of cell types; however, the limits of sensitivity of this potentially important phenomenon are unknown. Using the non-excitable rat megakaryocyte as a model system, we now show that P2Y receptor-evoked Ca2+ mobilization is controlled by membrane voltage in a graded and bipolar manner without evidence for a discrete threshold potential. Throughout the range of potentials studied, the peak increase in intracellular Ca2+ concentration ([Ca2+]i) in response to depolarization was always larger than the maximal reduction in [Ca2+]i following an equivalent amplitude hyperpolarization. Significant [Ca2+]i increases were observed in response to small amplitude (< 5 mV, 5 s duration) or short duration (25 ms, 135 mV) depolarizations. Individual cardiac action potential waveforms were also able to repeatedly potentiate P2Y receptor-evoked Ca2+ release and the response to trains of normally paced stimuli fused to generate prolonged [Ca2+]i increases. Furthermore, elevation of the temperature to physiological levels (36 degrees C) resulted in a more sustained depolarization-evoked Ca2+ increase compared with more transient or oscillatory responses at 20-24 degrees C. The ability of signalling via a G-protein-coupled receptor to be potentiated by action potential waveforms and small amplitude depolarizations has broad implications in excitable and non-excitable tissues.