Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics

Abstract The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.

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LUXendins reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics

10.1101/557132 ◽

2019 ◽

Author(s):

Julia Ast ◽

Anastasia Arvaniti ◽

Nicholas H.F. Fine ◽

Daniela Nasteska ◽

Fiona B. Ashford ◽

...

Keyword(s):

Genetic Manipulation ◽

Expression Patterns ◽

Receptor Activation ◽

Antibody Detection ◽

Fixed Tissue ◽

Incretin Mimetics ◽

Single Receptor ◽

Class B ◽

Glucagon Like Peptide ◽

Glucagon Like Peptide 1

ABSTRACTThe glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and STED-compatible LUXendin651 we describe islet GLP1R expression patterns, reveal higher-order GLP1R organization including the existence of membrane nanodomains, and track single receptor subpopulations. We furthermore show that different fluorophores can confer agonistic behavior on the LUXendin backbone, with implications for the design of stabilized incretin-mimetics. Thus, our labeling probes possess divergent activation modes, allow visualization of endogenous GLP1R, and provide new insight into class B GPCR distribution and dynamics.

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Abstract 146: Glucagon-like Peptide-1 Receptor Activation Promotes Dual Benefits For Reversing Microvasculopathy And Mitochondrial Damage In Diabetic Heart.

Circulation Research ◽

10.1161/res.113.suppl_1.a146 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Akio Monji ◽

Yasuko K Bando ◽

Toko Mitsui ◽

Morihiko Aoyama ◽

Hiroya Kawase ◽

...

Keyword(s):

Oxidative Stress ◽

Receptor Activation ◽

Left Ventricular ◽

Mitochondrial Damage ◽

Wall Thickening ◽

Mouse Heart ◽

Glucagon Like Peptide ◽

Glucagon Like Peptide 1 ◽

The Impact

PURPOSE: Glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (Ex4) is a remedy for type 2 diabetes mellitus (T2DM). Ex4 ameliorates cardiac dysfunction in preclinical and clinical settings. However, it remains unclear whether the impact of Ex4 on cardiac remodeling in diabetic cardiomyopathy (DMC), of which primary characteristics are microvasculopathy and mitochondrial damage. Methods and Results: Diet-induced T2DM (DIO) mice and age- and gender-matched lean control mice were allocated into EX4 (24 nmole/kg/day for 40 days; DIO-Ex4 and LEAN-Ex4) and vehicle groups (DIO-veh and LEAN-veh). We first confirmed the GLP-1R expression in every single chamber of mouse heart by immunoblotting and PCR. Ex4 treatment ameliorated both systemic and cardiac insulin resistance without affecting body weight in DIO. Cardiac capillary density of DIO-veh was reduced compared to those LEAN-veh, which were reversed by Ex4 treatment. Tube formation assay and immunoblot analysis using culture endothelial cells revealed that Ex4 directly enhanced in vitro angiogenesis in a PKA/eNOS-dependent fashion. Systolic and diastolic left-ventricular (LV) dysfunctions observed in DIO-veh were restored by Ex4 with decline in LV wall thickening. Myocardial fibrosis detected using sirius-red staining and tissue oxidative stress detected by a fluorescence indicator DHE were attenuated in DIO-Ex4. Of note, analyses using transmission electron microscopy and a fluorescence indicator for damaged mitochondria (mitotracker red) revealed that Ex4 treatment reversed cardiac mitochondrial remodeling and increased healthy mitochondria. Ex4 treatment modulated cardiac oxidative stress balance by upregulating antioxidative molecules (SOD, thioredoxin, glutathione peroxidase) and reduction of NOX4 level; whereas it had no influence on NOX2 level. Conclusions: Ex4 enhances cardiac angiogenesis via GLP-1R-mediated activation of PKA/eNOS axis and accelerates reverses remodeling of myocardial mitochondria, at least in part, via its facilitating effects on antioxidative defense.

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The Effects of a Weight-Loss Herbal Formula RCM-107 and Its Eight Individual Ingredients on Glucagon-Like Peptide-1 Secretion—An In Vitro and In Silico Study

International Journal of Molecular Sciences ◽

10.3390/ijms21082854 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2854 ◽

Cited By ~ 1

Author(s):

Shiqi Luo ◽

Harsharn Gill ◽

Bryce Feltis ◽

Andrew Hung ◽

Linh Toan Nguyen ◽

...

Keyword(s):

Weight Loss ◽

Active Form ◽

Receptor Activation ◽

Enzyme Linked Immunosorbent Assay ◽

Published Data ◽

Herbal Formula ◽

Receptor Agonists ◽

Glucagon Like Peptide ◽

Glucagon Like Peptide 1

Obesity is a multifactorial disease that can lead to other health issues. Glucagon-like peptide-1(GLP-1), as one of the satiety signal, has been linked with appetite suppression and weight loss. Due to the limitations of GLP-1 and its analogues, alternative treatments such as herbal therapies have become popular. The herbal formula RCM-107 has demonstrated its inhibitory effects on lipid and carbohydrate absorption in our previous work. However, no published data described its effects on GLP-1 secretion. Therefore, this study aimed to determine the effects of RCM-107 and its individual ingredients on GLP-1 secretion via enzyme-linked immunosorbent assay (ELISA). Furthermore, molecular docking was performed to predict the key chemical compounds that are likely to be GLP-1 receptor agonists. Gardeniae fructus, one of the ingredients in RCM-107, demonstrated significantly greater effects on inducing GLP-1 secretion than the positive control epigallocatechin gallate (EGCG). Two Gardeniae fructus ligands, 3-epioleanolic acid and crocin were predicted to bind to the active form of GLP-1 receptor at the binding pocket with residues known for the receptor activation, suggesting that they could potentially serve as receptor agonists. Overall, this study reported the effects of researched herbs on GLP-1 secretion and proposed two compounds that may be responsible for antiobesity via GLP-1 receptor activation.

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The effect of chronic hypothalamic-pituitary-adrenal axis activation on hypothalamic glucagon-like peptide-1 action in an animal model of depression and in vitro studies

Endocrine Abstracts ◽

10.1530/endoabs.49.gp154 ◽

2017 ◽

Author(s):

Jan Detka ◽

Anna Kurek ◽

Mateusz Kucharczyk ◽

Joanna Slusarczyk ◽

Katarzyna Glombik ◽

...

Keyword(s):

Animal Model ◽

Hypothalamic Pituitary Adrenal Axis ◽

In Vitro Studies ◽

Hypothalamic Pituitary Adrenal ◽

Adrenal Axis ◽

Animal Model Of Depression ◽

Glucagon Like Peptide ◽

Glucagon Like Peptide 1 ◽

Pituitary Adrenal Axis

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Faculty Opinions recommendation of Glucagon-Like Peptide-1 and Its Class B G Protein-Coupled Receptors: A Long March to Therapeutic Successes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726741818.793524566 ◽

2016 ◽

Author(s):

Peter R Bernstein

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

Class B ◽

Glucagon Like Peptide ◽

Glucagon Like Peptide 1 ◽

G Protein Coupled

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Synthesis and in vitro biological evaluation of new pyrimidines as glucagon-like peptide-1 receptor agonists

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/j.bmcl.2017.09.032 ◽

2017 ◽

Vol 27 (22) ◽

pp. 5071-5075 ◽

Cited By ~ 1

Author(s):

Shaikha S. AlNeyadi ◽

Abdu Adem ◽

Naheed Amer ◽

Alaa A. Salem ◽

Ibrahim M. Abdou

Keyword(s):

Biological Evaluation ◽

Receptor Agonists ◽

Glucagon Like Peptide ◽

Glucagon Like Peptide 1

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Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

Molecular Endocrinology ◽

10.1210/me.2011-1160 ◽

2011 ◽

Vol 25 (10) ◽

pp. 1804-1818 ◽

Cited By ~ 37

Author(s):

K. Coopman ◽

R. Wallis ◽

G. Robb ◽

A. J. H. Brown ◽

G. F. Wilkinson ◽

...

Keyword(s):

Ligand Binding ◽

Structural Model ◽

Transmembrane Domain ◽

Transmembrane Helix ◽

Receptor Activation ◽

Agonist Affinity ◽

Camp Generation ◽

N Terminus ◽

Glucagon Like Peptide ◽

Glucagon Like Peptide 1

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.

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A synthetic glucagon-like peptide-1 analog with improved plasma stability

Journal of Endocrinology ◽

10.1677/joe.0.1590093 ◽

1998 ◽

Vol 159 (1) ◽

pp. 93-102 ◽

Cited By ~ 44

Author(s):

U Ritzel ◽

U Leonhardt ◽

M Ottleben ◽

A Ruhmann ◽

K Eckart ◽

...

Keyword(s):

Amino Acid ◽

Plasma Insulin ◽

Rat Plasma ◽

Plasma Stability ◽

Terminal Amino Acid ◽

Terminal Amino ◽

Glucagon Like Peptide ◽

Glucagon Like Peptide 1

Glucagon-like peptide-1 (GLP-1) is the most potent endogenous insulin-stimulating hormone. In the present study the plasma stability and biological activity of a GLP-1 analog, [Ser]GLP-1(7-36)amide, in which the second N-terminal amino acid alanine was replaced by serine, was evaluated in vitro and in vivo. Incubation of GLP-1 with human or rat plasma resulted in degradation of native GLP-1(7-36)amide to GLP-1(9-36)amide, while [Ser]GLP-1(7-36)amide was not significantly degraded by plasma enzymes. Using glucose-responsive HIT-T15 cells, [Ser]GLP-1(7-36)amide showed strong insulinotropic activity, which was inhibited by the specific GLP-1 receptor antagonist exendin-4(9-39)amide. Simultaneous i.v. injection of [Ser]GLP-1(7-36)amide and glucose in rats induced a twofold higher increase in plasma insulin levels than unmodified GLP-1(7-36)amide with glucose and a fivefold higher increase than glucose alone. [Ser]GLP-1(7-36)amide induced a 1.5-fold higher increase in plasma insulin than GLP-1(7-36)amide when given 1 h before i.v. application of glucose. The insulinotropic effect of [Ser]GLP-1(7-36)amide was suppressed by i.v. application of exendin-4(9-39)amide. The present data demonstrate that replacement of the second N-terminal amino acid alanine by serine improves the plasma stability of GLP-1(7-36)amide. The insulinotropic action in vitro and in vivo was not impaired significantly by this modification.

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