Gwen Tolhurst

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.

Glucose Sensing in L Cells: A Primary Cell Study

Summary 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 α-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.

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.

Glutamine Triggers and Potentiates Glucagon-Like Peptide-1 Secretion by Raising Cytosolic Ca2+ and cAMP

The effects of chemical (DPP-4) inhibition and genetic reduction of DPP-4 activity on bone quality were studied in wild-type and ovariectomized mice.

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.

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.

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 (P2X1) and two members of the G-protein-coupled receptor family (P2Y1 and P2Y12). P2Y1 and P2Y12 receptors have firmly established roles in platelet activation during thrombosis and haemostasis, whereas the importance of the P2X1 receptor has been more controversial. However, recent studies have demonstrated that P2X1 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 P2X1 receptors in platelet activation, particularly under conditions of shear stress and thus during arterial thrombosis. This review discusses the background behind discovery of P2X1 receptors in platelets and their precursor cell, the megakaryocyte, and how signalling via these ion channels may participate in platelet activation.

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

The molecular identity of platelet Ca2+ entry pathways is controversial. Furthermore, the extent to which Ca2+‐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 Mg2+, 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 Ca2+ influx pathway. This study defines for the first time the principal TRP channels within the primary megakaryocyte, which represent candidates for Ca2+ influx pathways activated by a diverse range of stimuli in the platelet and megakaryocyte.

Electrical activity-triggered glucagon-like peptide-1 secretion from primary murine L-cells.

Non‐technical summary  Glucagon like peptide 1 (GLP‐1) based therapies are now widely used for the treatment of diabetes. The physiological source of the hormone is the intestinal L‐cell, and attempts to boost secretion have been hindered by difficulties in distinguishing these cells from their epithelial neighbours and our consequent limited understanding of their physiology. Using recently developed transgenic mice with fluorescently labelled L‐cells, we show that these cells are electrically active and use voltage‐gated ion channels to couple the presence of nutrients to the secretion of GLP‐1. We present the identification and characterisation of the ion channels. This improves our understanding of enteroendocrine physiology and will support therapeutic programmes aiming to target gut hormone secretion.

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.