Truncated Glucagon-like Peptide-1 and Exendin-4 α-Conotoxin pl14a Peptide Chimeras Maintain Potency and α-Helicity and Reveal Interactions Vital for cAMP Signaling in Vitro.

Glucagon-like peptide-1 (GLP-1) signaling through the glucagon-like peptide 1 receptor (GLP-1R) is a key regulator of normal glucose metabolism, and exogenous GLP-1R agonist therapy is a promising avenue for the treatment of type 2 diabetes mellitus. To date, the development of therapeutic GLP-1R agonists has focused on producing drugs with an extended serum half-life. This has been achieved by engineering synthetic analogs of GLP-1 or the more stable exogenous GLP-1R agonist exendin-4 (Ex-4). These synthetic peptide hormones share the overall structure of GLP-1 and Ex-4, with a C-terminal helical segment and a flexible N-terminal tail. Although numerous studies have investigated the molecular determinants underpinning GLP-1 and Ex-4 binding and signaling through the GLP-1R, these have primarily focused on the length and composition of the N-terminal tail or on how to modulate the helicity of the full-length peptides. Here, we investigate the effect of C-terminal truncation in GLP-1 and Ex-4 on the cAMP pathway. To ensure helical C-terminal regions in the truncated peptides, we produced a series of chimeric peptides combining the N-terminal portion of GLP-1 or Ex-4 and the C-terminal segment of the helix-promoting peptide α-conotoxin pl14a. The helicity and structures of the chimeric peptides were confirmed using circular dichroism and NMR, respectively. We found no direct correlation between the fractional helicity and potency in signaling via the cAMP pathway. Rather, the most important feature for efficient receptor binding and signaling was the C-terminal helical segment (residues 22-27) directing the binding of Phe(22) into a hydrophobic pocket on the GLP-1R.

Insulin secretion from beta cells in intact mouse islets is targeted towards the vasculature.

AIMS/HYPOTHESIS: We set out to test the hypothesis that insulin secretion from beta cells is targeted towards the vasculature. METHODS: The spatial location of granule fusion was identified by live-cell two-photon imaging of mouse pancreatic beta cells within intact islets, using sulforhodamine B labelling. Three-dimensional (3D) immunofluorescence of pancreatic slices was used to identify the location of proteins associated with neuronal synapses. RESULTS: We demonstrated an asymmetric, non-random, distribution of sites of insulin granule fusion in response to glucose and focal targeting of insulin granule secretion to the beta cell membrane facing the vasculature. 3D immunofluorescence of islets showed that structural proteins, such as liprin, piccolo and Rab2-interacting molecule, normally associated with neuronal presynaptic targeting, were present in beta cells and enriched at the vascular face. In contrast, we found that syntaxin 1A and synaptosomal-associated protein 25 kDa (SNAP25) were relatively evenly distributed across the beta cells. CONCLUSIONS/INTERPRETATION: Our results show that beta cells in situ, within intact islets, are polarised and target insulin secretion. This evidence for an 'endocrine synapse' has wide implications for our understanding of stimulus-secretion coupling in healthy islets and in disease.

Glucose principally regulates insulin secretion in mouse islets by controlling the numbers of granule fusion events per cell.

AIMS/HYPOTHESIS: In dispersed single beta cells the response of each cell to glucose is heterogeneous. In contrast, within an islet, cell-to-cell communication leads to glucose inducing a more homogeneous response. For example, increases in NAD(P)H and calcium are relatively uniform across the cells of the islet. These data suggest that secretion of insulin from single beta cells within an islet should also be relatively homogeneous. The aim of this study was to test this hypothesis by determining the glucose dependence of single-cell insulin responses within an islet. METHODS: Two-photon microscopy was used to detect the glucose-induced fusion of single insulin granules within beta cells in intact mouse islets. RESULTS: First, we validated our assay and showed that the measures of insulin secretion from whole islets could be explained by the time course and numbers of granule fusion events observed. Subsequent analysis of the patterns of granule fusion showed that cell recruitment is a significant factor, accounting for a fourfold increase from 3 to 20 mmol/l glucose. However, the major factor is the regulation of the numbers of granule fusion events within each cell, which increase ninefold over the range of 3 to 20 mmol/l glucose. Further analysis showed that two types of granule fusion event occur: 'full fusion' and 'kiss and run'. We show that the relative frequency of each type of fusion is independent of glucose concentration and is therefore not a factor in the control of insulin secretion. CONCLUSIONS/INTERPRETATION: Within an islet, glucose exerts its main effect through increasing the numbers of insulin granule fusion events within a cell.

Helixconstraints and amino acid substitution in GLP-1 increase cAMP and insulin secretion but not beta-arrestin 2 signaling.

Glucagon-like peptide (GLP-1) is an endogenous hormone that induces insulin secretion from pancreatic islets and modified forms are used to treat diabetes mellitus type 2. Understanding how GLP-1 interacts with its receptor (GLP-1R) can potentially lead to more effective drugs. Modeling and NMR studies of the N-terminus of GLP-1 suggest a ß-turn between residues Glu9-Phe12 and a kinked alpha helix between Val16-Gly37. N-terminal turn constraints attenuated binding affinity and activity (compounds 1-8). Lys-Asp (i, i+4) crosslinks in the middle and at the C-terminus increased alpha helicity and cAMP stimulation without much effect on binding affinity or beta-arrestin 2 recruitment (compounds 9-18). Strategic positioning of helix-inducing constraints and amino acid substitutions (Tyr16, Ala22) increased peptide helicity and produced ten-fold higher cAMP potency (compounds 19-28) over GLP-1(7-37)-NH2. The most potent cAMP activator (compound 23) was also the most potent inducer of insulin secretion.

Cyclic alpha-conotoxin peptidomimetic chimeras as potent GLP-1R agonists.

Type 2 diabetes mellitus (T2DM) results from compromised pancreatic ß-cell function, reduced insulin production, and lowered insulin sensitivity in target organs resulting in hyperglycemia. The GLP-1 hormone has two biologically active forms, GLP-1-(7-37) and GLP-1-(7-36)amide, which are equipotent at the glucagon-like peptide-1 receptor (GLP-1R). These peptides are central both to normal glucose metabolism and dysregulation in T2DM. Several structurally modified GLP-1 analogues are now approved drugs, and a number of other analogues are in clinical trials. None of these compounds is orally bioavailable and all require parenteral delivery. Recently, a number of smaller peptidomimetics containing 11-12 natural and unnatural amino acids have been identified that have similar insulin regulating profiles as GLP-1. The α-conotoxins are a class of disulfide rich peptide venoms isolated from cone snails, and are known for their highly constrained structures and resistance to enzymatic degradation. In this study, we examined whether 11-residue peptidomimetics incorporated into α-conotoxin scaffolds, forming monocyclic or bicyclic compounds constrained by disulfide bonds and/or backbone cyclization, could activate the GLP-1 receptor (GLP-1R). Several compounds showed potent (nanomolar) agonist activity at GLP-1R, as evaluated via cAMP signaling. In addition, HPLC retention times and in silico calculations suggested that mono- and bicyclic compounds had more favorable n-octanol/water partition coefficients according to the virtual partition coefficient model (vLogP), while maintaining a smaller radius of gyration compared to corresponding uncyclized peptidomimetics. Our findings suggest that cyclic peptidomimetics provide a potential avenue for future design of potent, compact ligands targeting GLP-1R and possessing improved physicochemical properties.

Short Hydrophobic Peptides with Cyclic Constraints Are Potent Glucagon-like Peptide-1 Receptor (GLP-1R) Agonists.

Cyclic constraints are incorporated into an 11-residue analogue of the N-terminus of glucagon-like peptide-1 (GLP-1) to investigate effects of structure on agonist activity. Cyclization through linking side chains of residues 2 and 5 or 5 and 9 produced agonists at nM concentrations in a cAMP assay. 2D NMR and CD spectra revealed an N-terminal ß-turn and a C-terminal helix that differentially influenced affinity and agonist potency. These structures can inform development of small molecule agonists of the GLP-1 receptor to treat type 2 diabetes.

Real-time measurement of F-actin remodelling during exocytosis using Lifeact-EGFP transgenic animals.

F-actin remodelling is essential for a wide variety of cell processes. It is important in exocytosis, where F-actin coats fusing exocytic granules. The purpose of these F-actin coats is unknown. They may be important in stabilizing the fused granules, they may play a contractile role and promote expulsion of granule content and finally may be important in endocytosis. To elucidate these functions of F-actin remodelling requires a reliable method to visualize F-actin dynamics in living cells. The recent development of Lifeact-EGFP transgenic animals offers such an opportunity. Here, we studied the characteristics of exocytosis in pancreatic acinar cells obtained from the Lifeact-EGFP transgenic mice. We show that the time-course of agonist-evoked exocytic events and the kinetics of each single exocytic event are the same for wild type and Lifeact-EGFP transgenic animals. We conclude that Lifeact-EGFP animals are a good model to study of exocytosis and reveal that F-actin coating is dependent on the de novo synthesis of F-actin and that development of actin polymerization occurs simultaneously in all regions of the granule. Our insights using the Lifeact-EGFP mice demonstrate that F-actin coating occurs after granule fusion and is a granule-wide event.