Beta cell specific probing with fluorescent exendin-4 is progressively reduced in type 2 diabetic mouse models

Lehtonen, Janne; Schäffer, Lauge; Rasch, Morten Grønbech; Hecksher‐Sørensen, Jacob; Ahnfelt-Rønne, Jonas

doi:10.1080/19382014.2015.1137415

Cited by 16 publications

(33 citation statements)

References 45 publications

(53 reference statements)

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“…Instead, using cell clusters plated onto coverslips, we could better discern LUXendin645 labeling, revealing GLP1R expression in 18 ± 6% of α-cells (Fig. 4d–f), higher than that shown before using antibodies 19,34 and reporter genes 7 . Notably, GLP1R-expressing α-cells tended to adjoin, whereas those without the receptor were next to β-cells.…”

Section: Resultsmentioning

confidence: 64%

“…Confirming previous findings, a majority (86 ± 3%) of β-cells were positive for LUXendin645 (Fig 4d-f). 7,19…”

Section: Resultsmentioning

confidence: 99%

“…Compared to present chemical biology approaches, LUXendins possess a number of advantages for GLP1R labeling, which generally rely on Exendin4(1-39) labeled with for instance Cy3, Cy5 or FITC. 19–21,30 Firstly, the use of an antagonist encourages receptor recycling back to the membrane and retains receptor at the cell surface, which likely increases detection capability. Secondly, the GLP1R is not activated, meaning that results can be interpreted in the absence of potentially confounding cell signaling, such as that expected with agonists.…”

Section: Discussionmentioning

confidence: 99%

“…Secondly, the GLP1R is not activated, meaning that results can be interpreted in the absence of potentially confounding cell signaling, such as that expected with agonists. 19 Thirdly, Cy5 occupies the far-red range, leading to less background fluorescence, increasing depth penetration due to reduced scatter, and avoiding the use of more phototoxic wavelengths. 39 Together, these desirable properties open up the possibility to image expression and trafficking of native GLP1R over extended periods of time, when LUXendins are used in conjunction with a PAM.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, classical fluorescent protein-fusions, 8 self-labeling suicide enzymes (SNAP-, CLIP-, and Halo-tag), 9–11 “click chemistry” 12,13 and fluorogenic probes 14–16 have provided unprecedented insight into the localization and distribution of their respective targets in living cells. In particular, current approaches for visualizing the GLP1R have so far relied on monoclonal antibodies (mAbs) directed against GLP1R epitopes, 17,18 or fluorescent analogues of Exendin4(1-39), 19–21 a stabilized form of GLP-1 and the basis for the incretin-mimetic class of drugs. Moreover, floxed mouse models exist in which Cre recombinase is driven by the Glp1r promoter, allowing labeling of GLP1R-expressing cells when crossed with reporter mice.…”

Section: Introductionmentioning

confidence: 99%

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

Arvaniti

et al. 2019

Preprint

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37 38 39 Word count: 3534 (excluding Abstract, Methods, References and Legends). 40 41 42 ABSTRACT (150 words) 43The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor 44(GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, 45 antibody detection or the use of probes that stimulate receptor activation. Herein, we present 46LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 47 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R 48 signaling can additionally be evoked when the receptor is allosterically modulated in the 49 presence of LUXendin645. Using LUXendin645 and STED-compatible LUXendin651, we 50 describe islet GLP1R expression patterns, reveal higher-order GLP1R organization including 51 the existence of membrane nanodomains, and track single receptor subpopulations. We 52 furthermore show that different fluorophores can confer agonistic behavior on the LUXendin 53 backbone, with implications for the design of stabilized incretin-mimetics. Thus, our labeling 54probes possess divergent activation modes, allow visualization of endogenous GLP1R, and 55provide new insight into class B GPCR distribution and dynamics. 56 57 RESULTS 102 Design and synthesis of LUXendin555, LUXendin651 and LUXendin645 103Ideally, a fluorescent probe to specifically visualize a biomolecule should have the following 104 characteristics: straightforward synthesis and easy accessibility, high solubility, relative small 105 size, high specificity and affinity, and a fluorescent moiety that exhibits photostability, 106brightness, (far-)red fluorescence with an additional two-photon cross-section. Moreover, the 107 probe should be devoid of biological effects when applied to live cells and show good or no 108 cell permeability, depending on its target localization. While some of these points were 109addressed in the past (vide infra), we set out to achieve this high bar by designing a highly 110 specific fluorescent GLP1R antagonist using TMR, Cy5 and SiR fluorophores. As no small 111 molecule antagonists for the GLP1R are known, we turned to Exendin4(9-39), a potent 112antagonistic scaffold amenable to modification (Fig. 1). 28 We used solid-phase peptide 113 synthesis (SPPS) to generate an S39C mutant, 29 which provides a C-terminal thiol handle 114for late-stage installation of different fluorophores. As such, TMR-, Cy5-and SiR-conjugated 115 versions were obtained by means of cysteine-maleimide chemistry, termed LUXendin555, 116LUXendin645, and LUXendin651, respectively (spectral properties are shown in Table 1, 117HPLC traces and HRMS characterization can be found in the SI) ( Fig. 1). 118

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Section: Resultsmentioning

confidence: 64%

“…Confirming previous findings, a majority (86 ± 3%) of β-cells were positive for LUXendin645 (Fig 4d-f). 7,19…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Arvaniti

et al. 2019

Preprint

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Exendin‐4 analogs in insulinoma theranostics

Jansen

Lith

Boss

et al. 2019

Labelled Comp Radiopharmac

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Insulinomas are neuroendocrine tumors arising from the pancreatic beta cells. Currently, surgical resection is the therapy of choice, and therefore, preoperative localization of insulinomas is essential. Nearly all insulinomas show overexpression of the glucagon‐like peptide‐1 receptor (GLP‐1R), and therefore, radiolabeled GLP‐1 peptide analog exendin‐4 can be used for diagnosis and preoperative localization with nuclear imaging. Here, we present an overview of the development and clinical implementation of exendin‐4–based tracers for single‐photon emission computed tomography (SPECT) and positron emission tomography (PET) imaging of insulinomas, and we address the potential use of this molecule for optical imaging. At last, we discuss the possibilities and pitfalls of the use of exendin‐4–based tracers for therapeutic applications such as peptide receptor radionuclide therapy (PRRT) or targeted photodynamic therapy (tPDT), giving a future outlook on the use of exendin‐4 in insulinoma theranostics.

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Development and characterisation of a novel glucagon like peptide-1 receptor antibody

Biggs

Liang²,

Naylor³

et al. 2017

Diabetologia

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Aims/hypothesis Glucagon like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion by binding to GLP-1 receptors (GLP1Rs) on pancreatic beta cells. GLP-1 mimetics are used in the clinic for the treatment of type 2 diabetes, but despite their therapeutic success, several clinical effects of GLP-1 remain unexplained at a mechanistic level, particularly in extrapancreatic tissues. The aim of this study was to generate and characterise a monoclonal antagonistic antibody for the GLP1R for use in vivo. Methods A naive phage display selection strategy was used to isolate single-chain variable fragments (ScFvs) that bound to GLP1R. The ScFv with the highest affinity, Glp1R0017, was converted into a human IgG1 and characterised further. In vitro antagonistic activity was assessed in a number of assays: a cAMP-based homogenous time-resolved fluorescence assay in GLP1R-overexpressing cell lines, a live cell cAMP imaging assay and an insulin secretion assay in INS-1 832/3 cells. Glp1R0017 was further tested in immunostaining of mouse pancreas, and the ability of Glp1R0017 to block GLP1R in vivo was assessed by both IPGTT and OGTT in C57/Bl6 mice.

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Beta cell specific probing with fluorescent exendin-4 is progressively reduced in type 2 diabetic mouse models

Cited by 16 publications

References 45 publications

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

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

Exendin‐4 analogs in insulinoma theranostics

Development and characterisation of a novel glucagon like peptide-1 receptor antibody

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