GLP-1 stimulates insulin secretion by PKC-dependent TRPM4 and TRPM5 activation.

Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the KATP channel blocker tolbutamide, and the L-type Ca(2+) channel blocker isradipine; however, depolarization was abolished by lowering extracellular Na(+). The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of Na(+)-permeable TRPM4 and TRPM5 channels by mobilization of intracellular Ca(2+) from thapsigargin-sensitive Ca(2+) stores. Concordantly, GLP-1 effects were negligible in Trpm4 or Trpm5 KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by β cells.We thank David Wiggins for excellent technical assistance. This work was supported by the Medical Research Council, Diabetes UK (to R. Ramracheya ), Oxford Biomedical Research Centre (to A. Tarasov), the Wellcome Trust (Senior Investigator Awards to A. Galione and P. Rorsman), the Warwick Impact Fund (to C. Weston and G. Ladds), the Biotechnology and Biological Sciences Research Council (to G. Ladds), the Knut and Alice Wallenberg Foundation (to P. Rorsman), and the Swedish Research Council (to P. Rorsman). The initial stages of M. Shigeto’s stay in Oxford were supported by a fellowship from Kawasaki Medical School.This is the final version of the article. It was first available from the American Society for Clinical Investigation via http://dx.doi.org/10.1172/JCI8197

Potentiation of TRPM5 ion channel activity reveals a novel pathway for the development of antidiabetic medication

In this thesis we explored the therapeutic potential of modulating TRPM5 ion channel activity in the context of high fat diet-induced diabetes in mice. TRPM5 is a monovalent selective cation channel that is activated by increases in intracellular calcium. The channel is functionally expressed in the type II taste receptor cells in the taste buds, where it is essential for the transduction of sweet, bitter and umami taste. TRPM5 expression was also shown in pancreatic beta-cells. Trpm5-/- mice have impaired insulin secretion and a prediabetic glucose metabolism. Therefore potentiation of TRPM5 might lead to increased taste perception and increased insulin secretion. At the start of this project no compounds were available that could accomplish this. In a first part of this study, we developed a thallium-based fluorescence screening method to detect TRPM5 modulating molecules in a high throughput. We found that ketoconazole and robustic acid (amongst others) are potent inhibitors of TRPM5 and confirmed their inhibiting effect with patch clamp experiments. We identified and describe the first known TRPM5 potentiators chloro-acetoxy-quinoline and exemestane. More interesting was the observation that glimepiride, an oral antidiabetic, was a potentiator of TRPM5 activity. This shows that TRPM5 is already a target in the treatment of diabetes, albeit unknown. A second part of the study focused on the interaction between steviol glycosides and TRPM5. We identified steviol glycosides, the sweet molecules in the stevia rebaudiana plant, as selective TRPM5 potentiators. We analyzed in detail the effects of stevioside and steviol on TRPM5. We showed that the overwhelming sweet taste of steviol glycosides can be explained by a potentiation of TRPM5 in the taste receptor cells. Furthermore, steviol on its own does not taste sweet, but it increases taste perception for sweet, bitter and umami compounds in a TRPM5-dependent way. We measured potentiation of TRPM5 currents in an overexpression system and in pancreatic beta-cells. We observed increased calcium oscillations upon application of stevioside in WT but not on Trpm5-/- mice. We finally analyzed the effect of stevioside administration in a high-fat diet induced diabetic mouse model. We found that stevioside increased the insulin secretion due to potentiation of TRPM5 in the beta-cells of the pancreas. This led to the prevention of severe hyperglycemia in these mice and effectively delayed the onset of diabetes. The effects of stevioside are mediated through TRPM5, as no e ects were observed in knockout animals. Taken together, i) our data con rm the essential role of TRPM5 in taste perception and insulin secretion, which was previously suggested from studies with knockout mice. ii) We show that TRPM5 is essential for the biological action of steviol and steviol glycosides iii) We show that potentiation of TRPM5 channel activity is an efficient strategy to prevent the development of type 2 diabetes, at least in mice and possibly also in humans.status: publishe

Stevia Sweetener Enhances Pancreatic Beta-Cell Function by Potentiating TRPM5 Channel Activity

Steviol glycosides as stevioside and rebaudioside A are natural, non-caloric sweet-tasting organic molecules, present in the extracts of the scrub plant Stevia rebaudiana, which are widely used as sweeteners in consumer foods and beverages. TRPM5 is a Ca2+-activated cation channel expressed in pancreatic β-cells. We show potentiation of TRPM5 with stevioside, rebaudioside A and their aglycon steviol. In this way, there is an enhanced glucose-induced insulin secretion in a Trpm5-dependent manner. The daily consumption of stevioside prevents the development of high-fat-diet-induced diabetic hyperglycaemia in wild-type mice, but not in Trpm5−/− mice. In isolated human pancreatic islets, there is a stevioside-induced potentiation of the intracellular calcium dynamics. This is indicative for increased insulin secretion, as is observed with mice. These results elucidate a molecular mechanism of action of steviol glycosides and identify TRPM5 as a potential target to prevent and treat type 2 diabetes.status: publishe

A Thallium-Based Screening Procedure to Identify Molecules That Modulate the Activity of Ca2+-Activated Monovalent Cation-Selective Channels

TRPM5 functions as a calcium-activated monovalent cation-selective ion channel and is expressed in a variety of cell types. Dysfunction of this type of channel has been recently implied in cardiac arrhythmias, diabetes, and other pathologies. Therefore, a growing interest has emerged to develop the pharmacology of these ion channels. We optimized a screening assay based on the thallium flux through the TRPM5 channel and a fluorescent thallium dye as a probe for channel activity. We show that this assay is capable of identifying molecules that inhibit or potentiate calcium-activated monovalent cation-selective ion channels.status: publishe

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer's disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes. There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.status: publishe

TRPM4

TRPM4 is a Ca(2+)-activated nonselective cation channel. The channel is activated by an increase of intracellular Ca(2+) and is regulated by several factors including temperature and Pi(4,5)P2. TRPM4 allows Na(+) entry into the cell upon activation, but is completely impermeable to Ca(2+). Unlike TRPM5, its closest relative in the transient receptor potential family, TRPM4 proteins are widely expressed in the body. Currents with properties that are reminiscent of TRPM4 have been described in a variety of tissues since the advent of the patch clamp technology, but their physiological role is only beginning to be clarified with the increasing characterization of knockout mouse models for TRPM4. Furthermore, mutations in the TRPM4 gene have been associated with cardiac conduction disorders in human patients. This review aims to overview the currently available data on the functional properties of TRPM4 and the current understanding of its physiological role in healthy and diseased tissue.status: publishe

Human islets contain a subpopulation of glucagon-like peptide-1 secreting alpha cells that is increased in type 2 diabetes

OBJECTIVES: Our study shows that glucagon-like peptide-1 (GLP-1) is secreted within human islets and may play an unexpectedly important paracrine role in islet physiology and pathophysiology. It is known that α cells within rodent and human pancreatic islets are capable of secreting GLP-1, but little is known about the functional role that islet-derived GLP-1 plays in human islets. METHODS: We used flow cytometry, immunohistochemistry, perifusions, and calcium imaging techniques to analyse GLP-1 expression and function in islets isolated from cadaveric human donors with or without type 2 diabetes. We also used immunohistochemistry to analyse GLP-1 expression within islets from pancreatic biopsies obtained from living donors. RESULTS: We have demonstrated that human islets secrete ∼50-fold more GLP-1 than murine islets and that ∼40% of the total human α cells contain GLP-1. Our results also confirm that dipeptidyl peptidase-4 (DPP4) is expressed in α cells. Sitagliptin increased GLP-1 secretion from cultured human islets but did not enhance glucose-stimulated insulin secretion (GSIS) in islets from non-diabetic (ND) or type 2 diabetic (T2D) donors, suggesting that β cell GLP-1 receptors (GLP-1R) may already be maximally activated. Therefore, we tested the effects of exendin-9, a GLP-1R antagonist. Exendin-9 was shown to reduce GSIS by 39% and 61% in ND islets and T2D islets, respectively. We also observed significantly more GLP-1+ α cells in T2D islets compared with ND islets obtained from cadaveric donors. Furthermore, GLP-1+ α cells were also identified in pancreatic islet sections obtained from living donors undergoing surgery. CONCLUSIONS: In summary, we demonstrated that human islets secrete robust amounts of GLP-1 from an α cell subpopulation and that GLP-1R signalling may support GSIS to a greater extent in T2D islets.status: publishe

TRPM4-dependent post-synaptic depolarization is essential for the induction of NMDA receptor-dependent LTP in CA1 hippocampal neurons

TRPM4 is a calcium-activated but calcium-impermeable non-selective cation (CAN) channel. Previous studies have shown that TRPM4 is an important regulator of Ca2+-dependent changes in membrane potential in excitable and non-excitable cell types. However, its physiological significance in neurons of the central nervous system remained unclear. Here, we report that TRPM4 proteins form a CAN channel in CA1 neurons of the hippocampus and we show that TRPM4 is an essential co-activator of N-methyl-D-aspartate (NMDA) receptors (NMDAR) during the induction of long-term potentiation (LTP). Disrupting the Trpm4 gene in mice specifically eliminates NMDAR-dependent LTP, while basal synaptic transmission, short-term plasticity, and NMDAR-dependent long-term depression are unchanged. The induction of LTP in Trpm4 -/- neurons was rescued by facilitating NMDA receptor activation or post-synaptic membrane depolarization. Accordingly, we obtained normal LTP in Trpm4 -/- neurons in a pairing protocol, where post-synaptic depolarization was applied in parallel to pre-synaptic stimulation. Taken together, our data are consistent with a novel model of LTP induction in CA1 hippocampal neurons, in which TRPM4 is an essential player in a feed-forward loop that generates the post-synaptic membrane depolarization which is necessary to fully activate NMDA receptors during the induction of LTP but which is dispensable for the induction of long-term depression (LTD). These results have important implications for the understanding of the induction process of LTP and the development of nootropic medication.status: publishe

Bax inhibitor-1 deficiency leads to obesity by increasing Ca2+-dependent insulin secretion

Transmembrane BAX inhibitor motif containing 6 (TMBIM6), also known as Bax inhibitor-1, is an evolutionarily conserved protein involved in endoplasmic reticulum (ER) function. TMBIM6 is an ER C