Naturally-occurring TGR5 agonists modulating glucagon-like peptide-1 biosynthesis and secretion

Selective GLP-1 secretagogues represent a novel potential therapy for type 2 diabetes mellitus. This study examined the GLP-1 secretory activity of the ethnomedicinal plant, Fagonia cretica, which is postulated to possess anti-diabetic activity. After extraction and fractionation extracts and purified compounds were tested for GLP-1 and GIP secretory activity in pGIP/neo STC-1 cells. Intracellular levels of incretin hormones and their gene expression were also determined. Crude F. cretica extracts stimulated both GLP-1 and GIP secretion, increased cellular hormone content, and upregulated gene expression of proglucagon, GIP and prohormone convertase. However, ethyl acetate partitioning significantly enriched GLP-1 secretory activity and this fraction underwent bioactivity-guided fractionation. Three isolated compounds were potent and selective GLP-1 secretagogues: quinovic acid (QA) and two QA derivatives, QA-3β-O-β-d-glycopyranoside and QA-3β-O-β-d-glucopyranosyl-(28 → 1)-β-d-glucopyranosyl ester. All QA compounds activated the TGR5 receptor and increased intracellular incretin levels and gene expression. QA derivatives were more potent GLP-1 secretagogues than QA. This is the first time that QA and its naturally-occurring derivatives have been shown to activate TGR5 and stimulate GLP-1 secretion. These data provide a plausible mechanism for the ethnomedicinal use of F. cretica and may assist in the ongoing development of selective GLP-1 agonists.     

Apelin stimulates both cholecystokinin and glucagon-like peptide 1 secretions in vitro and in vivo in rodents

Apelin is an enteric peptide that exerts several digestive functions such as stimulation of cell proliferation and cholecystokinin (CCK) secretion. We investigated using murine enteroendocrine cell line (STC-1) and rats if apelin-13 stimulates both CCK and glucagon-like peptide 1 (GLP-1) secretions. We demonstrated that, in vitro and in vivo, apelin-13 increases the release of these two hormones in a dose-dependent manner. Present data suggest that apelin may modulate digestive functions, food intake behavior and glucose homoeostasis via apelin-induced release of enteric CCK but also through a new incretin-releasing activity on enteric GLP-1.     

Bitter Taste Receptors Influence Glucose Homeostasis

TAS1R- and TAS2R-type taste receptors are expressed in the gustatory system, where they detect sweet- and bitter-tasting stimuli, respectively. These receptors are also expressed in subsets of cells within the mammalian gastrointestinal tract, where they mediate nutrient assimilation and endocrine responses. For example, sweeteners stimulate taste receptors on the surface of gut enteroendocrine L cells to elicit an increase in intracellular Ca²⁺ and secretion of the incretin hormone glucagon-like peptide-1 (GLP-1), an important modulator of insulin biosynthesis and secretion. Because of the importance of taste receptors in the regulation of food intake and the alimentary responses to chemostimuli, we hypothesized that differences in taste receptor efficacy may impact glucose homeostasis. To address this issue, we initiated a candidate gene study within the Amish Family Diabetes Study and assessed the association of taste receptor variants with indicators of glucose dysregulation, including a diagnosis of type 2 diabetes mellitus and high levels of blood glucose and insulin during an oral glucose tolerance test. We report that a TAS2R haplotype is associated with altered glucose and insulin homeostasis. We also found that one SNP within this haplotype disrupts normal responses of a single receptor, TAS2R9, to its cognate ligands ofloxacin, procainamide and pirenzapine. Together, these findings suggest that a functionally compromised TAS2R receptor negatively impacts glucose homeostasis, providing an important link between alimentary chemosensation and metabolic disease.     

Bile acids promote glucagon-like peptide-1 secretion through TGR5 in a murine enteroendocrine cell line STC-1

Bile acids play essential roles in the absorption of dietary lipids and in the regulation of bile acid biosynthesis. Recently, a G protein-coupled receptor, TGR5, was identified as a cell-surface bile acid receptor. In this study, we show that bile acids promote glucagon-like peptide-1 (GLP-1) secretion through TGR5 in a murine enteroendocrine cell line STC-1. In STC-1 cells, bile acids promoted GLP-1 secretion in a dose-dependent manner. As STC-1 cells express TGR5 mRNA, we examined whether bile acids induce GLP-1 secretion through TGR5. RNA interference experiments showed that reduced expression of TGR5 resulted in reduced secretion of GLP-1. Furthermore, transient transfection of STC-1 cells with an expression plasmid containing TGR5 significantly enhanced GLP-1 secretion, indicating that bile acids promote GLP-1 secretion through TGR5 in STC-1 cells. Bile acids induced rapid and dose-dependent elevation of intracellular cAMP levels in STC-1 cells. An adenylate cyclase inhibitor, MDL12330A, significantly suppressed bile acid-promoted GLP-1 secretion, suggesting that bile acids induce GLP-1 secretion via intracellular cAMP production in STC-1 cells.     

Osteocalcin Induces Release of Glucagon-Like Peptide-1 and Thereby Stimulates Insulin Secretion in Mice

The uncarboxylated form (ucOC), but not the γ-carboxylated form (GlaOC), of the bone-derived protein osteocalcin stimulates insulin secretion and regulates energy metabolism in insulin target tissues. Glucagon-like peptide–1 (GLP-1) is an insulin secretagogue that is released from the gut in response to food intake. We have now found that Gprc6a, a putative ucOC receptor, is expressed in epithelial cells of the mouse small intestine as well as in STC-1 enteroendocrine cells. Secretion of GLP-1 by STC-1 cells was stimulated by ucOC but not by GlaOC. The serum GLP-1 concentration in mice was increased by intraperitoneal or oral administration of ucOC, whereas GlaOC was effective in this regard only after oral application. Serum insulin levels were also increased by ucOC, and this effect was potentiated by an inhibitor of dipeptidyl peptidase IV and blocked by a GLP-1 receptor antagonist. Intravenous injection of ucOC in mice increased the serum GLP-1 concentration, and also increased the serum level of insulin. Our results suggest that ucOC acts via Gprc6a to induce GLP-1 release from the gut, and that the stimulatory effect of ucOC on insulin secretion is largely mediated by GLP-1.     

Molecular physiology of glucagon-like peptide-1 insulin secretagogue action in pancreatic β cells

Insulin secretion from pancreatic β cells is stimulated by glucagon-like peptide-1 (GLP-1), a blood glucose-lowering hormone that is released from enteroendocrine L cells of the distal intestine after the ingestion of a meal. GLP-1 mimetics (e.g., Byetta) and GLP-1 analogs (e.g., Victoza) activate the β cell GLP-1 receptor (GLP-1R), and these compounds stimulate insulin secretion while also lowering levels of blood glucose in patients diagnosed with type 2 diabetes mellitus (T2DM). An additional option for the treatment of T2DM involves the administration of dipeptidyl peptidase-IV (DPP-IV) inhibitors (e.g., Januvia, Galvus). These compounds slow metabolic degradation of intestinally released GLP-1, thereby raising post-prandial levels of circulating GLP-1 substantially. Investigational compounds that stimulate GLP-1 secretion also exist, and in this regard a noteworthy advance is the demonstration that small molecule GPR119 agonists (e.g., AR231453) stimulate L cell GLP-1 secretion while also directly stimulating β cell insulin release. In this review, we summarize what is currently known concerning the signal transduction properties of the β cell GLP-1R as they relate to insulin secretion. Emphasized are the cyclic AMP, protein kinase A, and Epac2-mediated actions of GLP-1 to regulate ATP-sensitive K⁺ channels, voltage-dependent K⁺ channels, TRPM2 cation channels, intracellular Ca²⁺ release channels, and Ca²⁺-dependent exocytosis. We also discuss new evidence that provides a conceptual framework with which to understand why GLP-1R agonists are less likely to induce hypoglycemia when they are administered for the treatment of T2DM.     

Investigating the effects of physiological bile acids on GLP-1 secretion and glucose tolerance in normal and GLP-1R(-/-) mice

Physiological secretion of bile acids has previously been linked to the regulation of blood glucose. GLP-1 is an intestinal peptide hormone with important glucose-lowering actions, such as stimulation of insulin secretion and inhibition of glucagon secretion. In this investigation, we assessed the ability of several bile acid compounds to secrete GLP-1 in vitro in STC-1 cells. Bile acids stimulated GLP-1 secretion from 3.3- to 6.2-fold but some were associated with cytolytic effects. Glycocholic and taurocholic acids were selected for in vivo studies in normal and GLP-1R(-/-) mice. Oral glucose tolerance tests revealed that glycocholic acid did not affect glucose excursions. However, taurocholic acid reduced glucose excursions by 40% in normal mice and by 27% in GLP-1R(-/-) mice, and plasma GLP-1 concentrations were significantly elevated 30 min post-gavage. Additional studies used incretin receptor antagonists to probe involvement of GLP-1 and GIP in taurocholic acid-induced glucose lowering. The findings suggest that bile acids partially aid glucose regulation by physiologically enhancing nutrient-induced GLP-1 secretion. However, GLP-1 secretion appears to be only part of the glucose-lowering mechanism and our studies indicate that the other major incretin GIP is not involved.     

Involvement of the Calcium-sensing Receptor in Human Taste Perception

By human sensory analyses, we found that various extracellular calcium-sensing receptor (CaSR) agonists enhance sweet, salty, and umami tastes, although they have no taste themselves. These characteristics are known as “kokumi taste” and often appear in traditional Japanese cuisine. Although GSH is a typical kokumi taste substance (taste enhancer), its mode of action is poorly understood. Here, we demonstrate how the kokumi taste is enhanced by the CaSR, a close relative of the class C G-protein-coupled receptors T1R1, T1R2, and T1R3 (sweet and umami receptors). We identified a large number of CaSR agonist γ-glutamyl peptides, including GSH (γ-Glu-Cys-Gly) and γ-Glu-Val-Gly, and showed that these peptides elicit the kokumi taste. Further analyses revealed that some known CaSR agonists such as Ca²⁺, protamine, polylysine, l-histidine, and cinacalcet (a calcium-mimetic drug) also elicit the kokumi taste and that the CaSR-specific antagonist, NPS-2143, significantly suppresses the kokumi taste. This is the first report indicating a distinct function of the CaSR in human taste perception.     

The effect of encapsulated glutamine on gut peptide secretion in human volunteers

ContextWeight loss and improved blood glucose control after bariatric surgery have been attributed in part to increased ileal nutrient delivery with enhanced release of glucagon-like peptide 1 (GLP-1). Non-surgical strategies to manage obesity are required. The aim of the current study was to assess whether encapsulated glutamine, targeted to the ileum, could increase GLP-1 secretion, improve glucose tolerance or reduce meal size.MethodsA single-center, randomised, double blind, placebo-controlled, cross-over study was performed in 24 healthy volunteers and 8 patients with type 2 diabetes. Fasting participants received a single dose of encapsulated ileal-release glutamine (3.6 or 6.0 g) or placebo per visit with blood sampling at baseline and for 4 h thereafter. Glucose tolerance and meal size were studied using a 75 g oral glucose tolerance test and ad libitum meal respectively.ResultsIn healthy volunteers, ingestion of 6.0 g glutamine was associated with increased GLP-1 concentrations after 90 min compared with placebo (mean 10.6 pg/ml vs 6.9 pg/ml, p = 0.004), increased insulin concentrations after 90 min (mean 70.9 vs 48.5, p = 0.048), and increased meal size at 120 min (mean 542 g eaten vs 481 g, p = 0.008). Ingestion of 6.0 g glutamine was not associated with significant differences in GLP-1, glucose or insulin concentrations after a glucose tolerance test in healthy or type 2 diabetic participants.ConclusionsSingle oral dosing of encapsulated glutamine did not provoke consistent increases in GLP-1 and insulin secretion and was not associated with beneficial metabolic effects in healthy volunteers or patients with type 2 diabetes.     

Novel GLP-1 Fusion Chimera as Potent Long Acting GLP-1 Receptor Agonist

GLP-1 has a variety of anti-diabetic effects. However, native GLP-1 is not suitable for therapy of diabetes due to its short half-life (t1/2<2 min). To circumvent this, we developed a long-lasting GLP-1 receptor agonist by the fusion of GLP-1 with human IgG2 Fc (GLP-1/hIgG2). ELISA-based receptor binding assay demonstrated that GLP-1/hIgG2 had high binding affinity to the GLP-1R in INS-1 cells (Kd = 13.90±1.52 nM). Upon binding, GLP-1/hIgG2 was rapidly internalized by INS-1 cells in a dynamin-dependent manner. Insulin RIA showed that GLP-1/IgG2 dose-dependently stimulated insulin secretion from INS-1 cells. Pharmacokinetic studies in CD1 mice showed that with intraperitoneal injection (i.p.), the GLP-1/hIgG2 peaked at 30 minutes in circulation and maintained a plateau for >168 h. Intraperitoneal glucose tolerance test (IPGTT) in mice showed that GLP-1/hIgG2 significantly decreased glucose excursion. Furthermore, IPGTT performed on mice one week after a single drug-injection also displayed significantly reduced glucose excursion, indicating that GLP-1/hIgG2 fusion protein has long-lasting effects on the modulation of glucose homeostasis. GLP-1/hIgG2 was found to be effective in reducing the incidence of diabetes in multiple-low-dose streptozotocin-induced type 1 diabetes in mice. Together, the long-lasting bioactive GLP-1/hIgG2 retains native GLP-1 activities and thus may serve as a potent GLP-1 receptor agonist.     

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 Ca²⁺ 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.     

A Novel Glucagon-like Peptide-1 (GLP-1)/Glucagon Hybrid Peptide with Triple-acting Agonist Activity at Glucose-dependent Insulinotropic Polypeptide,GLP-1, and Glucagon Receptors and Therapeutic Potential in High Fat-fed Mice

Glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon bind to related members of the same receptor superfamily and exert important effects on glucose homeostasis, insulin secretion, and energy regulation. The present study assessed the biological actions and therapeutic utility of novel GIP/glucagon/GLP-1 hybrid peptides. Nine novel peptides were synthesized and exhibited complete DPP-IV resistance and enhanced in vitro insulin secretion. The most promising peptide, [dA²]GLP-1/GcG, stimulated cAMP production in GIP, GLP-1, and glucagon receptor-transfected cells. Acute administration of [dA²]GLP-1/GcG in combination with glucose significantly lowered plasma glucose and increased plasma insulin in normal and obese diabetic (ob/ob) mice. Furthermore, [dA²]GLP-1/GcG elicited a protracted glucose-lowering and insulinotropic effect in high fat-fed mice. Twice daily administration of [dA²]GLP-1/GcG for 21 days decreased body weight and nonfasting plasma glucose and increased circulating plasma insulin concentrations in high fat-fed mice. Furthermore, [dA²]GLP-1/GcG significantly improved glucose tolerance and insulin sensitivity by day 21. Interestingly, locomotor activity was increased in [dA²]GLP-1/GcG mice, without appreciable changes in aspects of metabolic rate. Studies in knock-out mice confirmed the biological action of [dA²]GLP-1/GcG via multiple targets including GIP, GLP-1, and glucagon receptors. The data suggest significant promise for novel triple-acting hybrid peptides as therapeutic options for obesity and diabetes.     

The G Protein-coupled Receptor Family C Group 6 Subtype A (GPRC6A) Receptor Is Involved in Amino Acid-induced Glucagon-like Peptide-1 Secretion from GLUTag Cells

Although amino acids are dietary nutrients that evoke the secretion of glucagon-like peptide 1 (GLP-1) from intestinal L cells, the precise molecular mechanism(s) by which amino acids regulate GLP-1 secretion from intestinal L cells remains unknown. Here, we show that the G protein-coupled receptor (GPCR), family C group 6 subtype A (GPRC6A), is involved in amino acid-induced GLP-1 secretion from the intestinal L cell line GLUTag. Application of l-ornithine caused an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in GLUTag cells. Application of a GPRC6A receptor antagonist, a phospholipase C inhibitor, or an IP₃ receptor antagonist significantly suppressed the l-ornithine-induced [Ca²⁺]_i increase. We found that the increase in [Ca²⁺]_i stimulated by l-ornithine correlated with GLP-1 secretion and that l-ornithine stimulation increased exocytosis in a dose-dependent manner. Furthermore, depletion of endogenous GPRC6A by a specific small interfering RNA (siRNA) inhibited the l-ornithine-induced [Ca²⁺]_i increase and GLP-1 secretion. Taken together, these findings suggest that the GPRC6A receptor functions as an amino acid sensor in GLUTag cells that promotes GLP-1 secretion.     

Lipid derivatives activate GPR119 and trigger GLP-1 secretion in primary murine L-cells

Aims/hypothesisGlucagon-like peptide-1 (GLP-1) is an incretin hormone derived from proglucagon, which is released from intestinal L-cells and increases insulin secretion in a glucose dependent manner. GPR119 is a lipid derivative receptor present in L-cells, believed to play a role in the detection of dietary fat. This study aimed to characterize the responses of primary murine L-cells to GPR119 agonism and assess the importance of GPR119 for the detection of ingested lipid.MethodsGLP-1 secretion was measured from murine primary cell cultures stimulated with a panel of GPR119 ligands. Plasma GLP-1 levels were measured in mice lacking GPR119 in proglucagon-expressing cells and controls after lipid gavage. Intracellular cAMP responses to GPR119 agonists were measured in single primary L-cells using transgenic mice expressing a cAMP FRET sensor driven by the proglucagon promoter.ResultsL-cell specific knockout of GPR119 dramatically decreased plasma GLP-1 levels after a lipid gavage. GPR119 ligands triggered GLP-1 secretion in a GPR119 dependent manner in primary epithelial cultures from the colon, but were less effective in the upper small intestine. GPR119 agonists elevated cAMP in ∼70% of colonic L-cells and 50% of small intestinal L-cells.Conclusions/interpretationGPR119 ligands strongly enhanced GLP-1 release from colonic cultures, reflecting the high proportion of colonic L-cells that exhibited cAMP responses to GPR119 agonists. Less GPR119-dependence could be demonstrated in the upper small intestine. In vivo, GPR119 in L-cells plays a key role in oral lipid-triggered GLP-1 secretion.     

Spatiotemporal Modeling of Triggering and Amplifying Pathways in GLP-1 Secreting Intestinal L Cells

Glucagon-like peptide 1 (GLP-1) is secreted by intestinal L-cells, and augments glucose-induced insulin secretion, thus playing an important role in glucose control. The stimulus-secretion pathway in L-cells is still incompletely understood and a topic of debate. It is known that GLP-1 secreting cells can sense glucose to promote electrical activity either by the electrogenic sodium-glucose cotransporter SGLT1, or by closure of ATP-sensitive potassium channels after glucose metabolism. Glucose also has an effect on GLP-1 secretion downstream of electrical activity. An important aspect to take into account is the spatial organization of the cell. Indeed, the glucose transporter GLUT2 is located at the basolateral, vascular side, while SGLT1 is exposed to luminal glucose at the apical side of the cell, suggesting that the two types of transporters play different roles in glucose sensing. Here, we extend our recent model of electrical activity in primary L-cells to include spatiotemporal glucose and Ca²⁺ dynamics, and GLP-1 secretion. The model confirmed that glucose transportation into the cell through SGLT1 cotransporters can induce Ca²⁺ influx and release of GLP-1 as a result of electrical activity, while glucose metabolism alone is insufficient to depolarize the cell and evoke GLP-1 secretion in the model, suggesting a crucial role for SGLT1 in triggering GLP-1 release in agreement with experimental studies. We suggest a secondary, but participating, role of GLUT2 and glucose metabolism for GLP-1 secretion via an amplifying pathway that increases the secretion rate at a given Ca²⁺ level.     

Oleoyl-lysophosphatidylinositol enhances glucagon-like peptide-1 secretion from enteroendocrine L-cells through GPR119

The gastrointestinal tract is increasingly viewed as critical in controlling glucose metabolism, because of its role in secreting multiple glucoregulatory hormones, such as glucagon like peptide-1 (GLP-1). Here we investigate the molecular pathways behind the GLP-1- and insulin-secreting capabilities of a novel GPR119 agonist, Oleoyl-lysophosphatidylinositol (Oleoyl-LPI). Oleoyl-LPI is the only LPI species able to potently stimulate the release of GLP-1 in vitro, from murine and human L-cells, and ex-vivo from murine colonic primary cell preparations. Here we show that Oleoyl-LPI mediates GLP-1 secretion through GPR119 as this activity is ablated in cells lacking GPR119 and in colonic primary cell preparation from GPR119^?/? mice. Similarly, Oleoyl-LPI-mediated insulin secretion is impaired in islets isolated from GPR119^?/? mice. On the other hand, GLP-1 secretion is not impaired in cells lacking GPR55 in vitro or in colonic primary cell preparation from GPR55^?/? mice. We therefore conclude that GPR119 is the Oleoyl-LPI receptor, upstream of ERK1/2 and cAMP/PKA/CREB pathways, where primarily ERK1/2 is required for GLP-1 secretion, while CREB activation appears dispensable.