Molecular Imaging and Biology

, Volume 18, Issue 1, pp 79–89 | Cite as

Quantitative Impact of Plasma Clearance and Down-regulation on GLP-1 Receptor Molecular Imaging

  • Liang Zhang
  • Greg M. ThurberEmail author
Research Article



Quantitative molecular imaging of beta cell mass (BCM) would enable early detection and treatment monitoring of type 1 diabetes. The glucagon-like peptide-1 (GLP-1) receptor is an attractive target due to its beta cell specificity and cell surface location. We quantitatively investigated the impact of plasma clearance and receptor internalization on targeting efficiency in healthy B6 mice.


Four exenatide-based probes were synthesized that varied in molecular weight, binding affinity, and plasma clearance. The GLP-1 receptor internalization rate and in vivo receptor expression were quantified.


Receptor internalization (54,000 receptors/cell in vivo) decreased significantly within minutes, reducing the benefit of a slower-clearing agent. The multimers and albumin binding probes had higher kidney and liver uptake, respectively.


Slow plasma clearance is beneficial for GLP-1 receptor peptide therapeutics. However, for exendin-based imaging of islets, down-regulation of the GLP-1 receptor and non-specific background uptake result in a higher target-to-background ratio for fast-clearing agents.

Key words

Exendin Glucagon-like peptide-1 receptor Type 1 diabetes imaging 



Funding was provided by NIH grant 1K01DK093766 (GMT). We thank Dr. Tim Scott and Tao Wei for assistance with NMR data, and Dr. Allen Liu for use of the spinning disk confocal microscope.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

11307_2015_880_MOESM1_ESM.pdf (2.2 mb)
ESM 1 (PDF 2203 kb) (4.3 mb)
ESM 2 (MOV 4427 kb)


  1. 1.
    Reiner T, Kohler RH, Liew CW et al (2010) Near-infrared fluorescent probe for imaging of pancreatic beta cells. Bioconjug Chem 21:1362–1368PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Bandara N, Zheleznyak A, Cherukuri K, et al. (2015) Evaluation of Cu-64 and Ga-68 radiolabeled glucagon-like peptide-1 receptor agonists as PET tracers for pancreatic beta cell imaging. Mol Imaging BiolGoogle Scholar
  3. 3.
    Goke B (2010) What are the potential benefits of clinical beta-cell imaging in diabetes mellitus? Curr Pharm Des 16:1547–1549CrossRefPubMedGoogle Scholar
  4. 4.
    Holmberg D, Ahlgren U (2008) Imaging the pancreas: from ex vivo to non-invasive technology. Diabetologia 51:2148–2154CrossRefPubMedGoogle Scholar
  5. 5.
    Arifin DR, Bulte JWM (2011) Imaging of pancreatic islet cells. Diabetes Metab Res Rev 27:761–766PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Ichise M, Harris PE (2010) Imaging of beta-cell mass and function. J Nucl Med 51:1001–1004PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Boerman OC, Gotthardt M (2012) F-18-Labelled exendin to image GLP-1 receptor-expressing tissues: from niche to blockbuster? Eur J Nucl Med Mol Imaging 39:461–462PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Foulis AK, Liddle CN, Farquharson MA, Richmond JA, Weir RS (1986) The histopathology of the pancreas in type 1 (insulin-dependent) diabetes mellitus: a 25-year review of deaths in patients under 20 years of age in the United Kingdom. Diabetologia 29:267–274CrossRefPubMedGoogle Scholar
  9. 9.
    Chmelova H, Cohrs CM, Chouinard JA et al (2015) Distinct roles of beta cell mass and function during type 1 diabetes onset and remission. Diabetes 64:2148–2160CrossRefPubMedGoogle Scholar
  10. 10.
    Saudek F, Brogren CH, Manohar S (2008) Imaging the beta-cell mass: why and how. Rev Diabet Stud 5:6–12PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Steiner DJ, Kim A, Miller K, Hara M (2010) Pancreatic islet plasticity interspecies comparison of islet architecture and composition. Islets 2:135–145PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Saisho Y, Butler AE, Manesso E et al (2013) Beta-cell mass and turnover in humans effects of obesity and aging. Diabetes Care 36:111–117PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Medarova Z, Evgenov NV, Dai G et al (2006) In vivo multimodal imaging of transplanted pancreatic islets. Nat Protoc 1:429–435CrossRefPubMedGoogle Scholar
  14. 14.
    McCulloch DK, Koerker DJ, Kahn SE et al (1991) Correlations of in vivo beta-cell function tests with beta-cell mass and pancreatic insulin content in streptozocin-administered baboons. Diabetes 40:673–679CrossRefPubMedGoogle Scholar
  15. 15.
    Brom M, Andraojc K, Oyen WJG et al (2010) Development of radiotracers for the determination of the beta-cell mass in vivo. Curr Pharm Des 16:1561–1567CrossRefPubMedGoogle Scholar
  16. 16.
    Goland R, Freeby M, Parsey R et al (2009) 11C-dihydrotetrabenazine PET of the pancreas in subjects with long-standing type 1 diabetes and in healthy controls. J Nucl Med 50:382–389PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Gepts W, Lecompte PM (1981) The pancreatic islets in diabetes. Am J Med 70:105–115CrossRefPubMedGoogle Scholar
  18. 18.
    Sweet IR, Cook DL, Lernmark A et al (2004) Systematic screening of potential beta-cell imaging agents. Biochem Biophys Res Commun 314:976–983CrossRefPubMedGoogle Scholar
  19. 19.
    Moore A, Bonner-Weir S, Weissleder R (2001) Noninvasive in vivo measurement of beta-cell mass in mouse model of diabetes. Diabetes 50:2231–2236CrossRefPubMedGoogle Scholar
  20. 20.
    Schneider S, Feilen PJ, Schreckenberger M et al (2005) In vitro and in vivo evaluation of novel glibenclamide derivatives as imaging agents for the non-invasive assessment of the pancreatic islet cell mass in animals and humans. Exp Clin Endocrinol Diabetes 113:388–395CrossRefPubMedGoogle Scholar
  21. 21.
    Harris PE, Ferrara C, Barba P et al (2008) VMAT2 gene expression and function as it applies to imaging beta-cell mass. J Mol Med 86:5–16CrossRefPubMedGoogle Scholar
  22. 22.
    Souza F, Simpson N, Raffo A et al (2006) Longitudinal noninvasive PET-based beta cell mass estimates in a spontaneous diabetes rat model. J Clin Invest 116:1506–1513PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Virostko J, Henske J, Vinet L et al (2011) Multimodal image coregistration and inducible selective cell ablation to evaluate imaging ligands. Proc Natl Acad Sci U S A 108:20719–20724PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Reiner T, Thurber G, Gaglia J et al (2011) Accurate measurement of pancreatic islet beta-cell mass using a second-generation fluorescent exendin-4 analog. Proc Natl Acad Sci U S A 108:12815–12820PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    Di Gialleonardo V, de Vries EF, Di Girolamo M et al (2012) Imaging of beta-cell mass and insulitis in insulin-dependent (type 1) diabetes mellitus. Endocr Rev 33:892–919CrossRefPubMedGoogle Scholar
  26. 26.
    Basu S, Alavi A (2007) Partial volume correction of standardized uptake values and the dual time point in FDG-PET imaging: should these be routinely employed in assessing patients with cancer? Eur J Nucl Med Mol Imaging 34:1527–1529CrossRefPubMedGoogle Scholar
  27. 27.
    Basu S, Zaidi H, Houseni M et al (2007) Novel quantitative techniques for assessing regional and global function and structure based on modern imaging modalities: implications for normal variation, aging and diseased states. Semin Nucl Med 37:223–239CrossRefPubMedGoogle Scholar
  28. 28.
    Chawluk JB, Alavi A, Dann R et al (1987) Positron emission tomography in aging and dementia: effect of cerebral atrophy. J Nucl Med 28:431–437PubMedGoogle Scholar
  29. 29.
    Hickeson M, Yun MJ, Matthies A et al (2002) Use of a corrected standardized uptake value based on the lesion size on CT permits accurate characterization of lung nodules on FDG-PET. Eur J Nucl Med Mol Imaging 29:1639–1647CrossRefPubMedGoogle Scholar
  30. 30.
    Srinivas SM, Dhurairaj T, Basu S et al (2009) A recovery coefficient method for partial volume correction of PET images. Ann Nucl Med 23:341–348CrossRefPubMedGoogle Scholar
  31. 31.
    Tanna NK, Kohn MI, Horwich DN et al (1991) Analysis of brain and cerebrospinal-fluid volumes with MR imaging: impact on PET data correction for atrophy. Part II. Aging and Alzheimer dementia. Radiology 178:123–130CrossRefPubMedGoogle Scholar
  32. 32.
    Sweet IR, Cook DL, Lernmark A et al (2004) Non-invasive imaging of beta cell mass: a quantitative analysis. Diabetes Technol Ther 6:652–659CrossRefPubMedGoogle Scholar
  33. 33.
    Keliher EJ, Reiner T, Thurber GM et al (2012) Efficient 18F-labeling of synthetic exendin-4 analogues for imaging beta cells. ChemistryOpen 1:177–183PubMedCentralCrossRefPubMedGoogle Scholar
  34. 34.
    Brand C, Abdel-Atti D, Zhang Y et al (2014) In vivo imaging of GLP-1R with a targeted bimodal PET/fluorescence imaging agent. Bioconjug Chem 25:1323–1330PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Eng J, Kleinman WA, Singh L et al (1992) Isolation and characterization of exendin-4, an exendin-3 analog, from Heloderma suspectum venom. Further evidence for an exendin receptor on dispersed acini from guinea pig pancreas. J Biol Chem 267:7402–7405PubMedGoogle Scholar
  36. 36.
    Gotthardt M, Lalyko G, van Eerd-Vismale J et al (2006) A new technique for in vivo imaging of specific GLP-1 binding sites: first results in small rodents. Regul Pept 137:162–167CrossRefPubMedGoogle Scholar
  37. 37.
    Goke R, Fehmann HC, Linn T et al (1993) Exendin-4 is a high potency agonist and truncated exendin-(9–39)-amide an antagonist at the glucagon-like peptide 1-(7–36)-amide receptor of insulin-secreting beta-cells. J Biol Chem 268:19650–19655PubMedGoogle Scholar
  38. 38.
    Kolligs F, Fehmann HC, Goke R, Goke B (1995) Reduction of the incretin effect in rats by the glucagon-like peptide-1 receptor antagonist exendin(9–39) amide. Diabetes 44:16–19CrossRefPubMedGoogle Scholar
  39. 39.
    Gotthardt M, Fischer M, Naeher I et al (2002) Use of the incretin hormone glucagon-like peptide-1 (GLP-1) for the detection of insulinomas: initial experimental results. Eur J Nucl Med Mol Imaging 29:597–606CrossRefPubMedGoogle Scholar
  40. 40.
    Wicki A, Wild D, Storch D et al (2007) [Lys(40)(Ahx-DTPA-In-111)NH2]-Exendin-4 is a highly efficient radiotherapeutic for glucagon-like peptide-1 receptor-targeted therapy for insulinoma. Clin Cancer Res 13:3696–3705CrossRefPubMedGoogle Scholar
  41. 41.
    Mikkola K, Yim CB, Fagerholm V et al (2014) 64Cu- and 68Ga-labelled [Nle(14), Lys(40)(Ahx-NODAGA)NH2]-exendin-4 for pancreatic beta cell imaging in rats. Mol Imaging Biol 16:255–263CrossRefPubMedGoogle Scholar
  42. 42.
    Wild D, Wicki A, Mansi R et al (2010) Exendin-4-based radiopharmaceuticals for glucagonlike peptide-1 receptor PET/CT and SPECT/CT. J Nucl Med 51:1059–1067CrossRefPubMedGoogle Scholar
  43. 43.
    Zhang L, Navaratna T, Liao JS, Thurber GM (2015) Dual-purpose linker for alpha helix stabilization and imaging agent conjugation to glucagon-like peptide-1 receptor ligands. Bioconjug Chem 26:329–337CrossRefPubMedGoogle Scholar
  44. 44.
    Gao HK, Niu G, Yang M et al (2011) PET of insulinoma using F-18-FBEM-EM3106B, a new GLP-1 analogue. Mol Pharm 8:1775–1782PubMedCentralCrossRefPubMedGoogle Scholar
  45. 45.
    Virostko J, Jansen ED, Powers AC (2006) Current status of imaging pancreatic islets. Curr Diabetes Rep 6:328–332CrossRefGoogle Scholar
  46. 46.
    Kim TH, Jiang HH, Lee S et al (2011) Mono-PEGylated dimeric exendin-4 as high receptor binding and long-acting conjugates for type 2 anti-diabetes therapeutics. Bioconjug Chem 22:625–632CrossRefPubMedGoogle Scholar
  47. 47.
    Kim TH, Jiang HH, Lim SM et al (2012) Site-specific PEGylated exendin-4 modified with a high molecular weight trimeric PEG reduces steric hindrance and increases type 2 antidiabetic therapeutic effects. Bioconjug Chem 23:2214–2220CrossRefPubMedGoogle Scholar
  48. 48.
    Widmann C, Dolci W, Thorens B (1995) Agonist-induced internalization and recycling of the glucagon-like peptide-1 receptor in transfected fibroblasts and in insulinomas. Biochem J 310(Pt 1):203–214PubMedCentralCrossRefPubMedGoogle Scholar
  49. 49.
    Schmidt MM, Thurber GM, Wittrup KD (2008) Kinetics of anti-carcinoembryonic antigen antibody internalization: effects of affinity, bivalency, and stability. Cancer Immunol Immunother 57:1879–1890PubMedCentralCrossRefPubMedGoogle Scholar
  50. 50.
    Oliveira S, Cohen R, Walsum MS et al (2012) A novel method to quantify IRDye800CW fluorescent antibody probes ex vivo in tissue distribution studies. EJNMMI Res 2:50PubMedCentralCrossRefPubMedGoogle Scholar
  51. 51.
    Runge S, Thogersen H, Madsen K et al (2008) Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain. J Biol Chem 283:11340–11347CrossRefPubMedGoogle Scholar
  52. 52.
    Berezin MY, Guo K, Akers W et al (2011) Rational approach to select small peptide molecular probes labeled with fluorescent cyanine dyes for in vivo optical imaging. Biochemistry 50:2691–2700PubMedCentralCrossRefPubMedGoogle Scholar
  53. 53.
    Thurber GM, Weissleder R (2011) A systems approach for tumor pharmacokinetics. PLoS ONE 6Google Scholar
  54. 54.
    Bhatnagar S, Deschenes E, Liao JS et al (2014) Multichannel imaging to quantify four classes of pharmacokinetic distribution in tumors. J Pharm Sci 103:3276–3286PubMedCentralCrossRefPubMedGoogle Scholar
  55. 55.
    Fujita H, Morii T, Fujishima H et al (2014) The protective roles of GLP-1R signaling in diabetic nephropathy: possible mechanism and therapeutic potential. Kidney Int 85:579–589CrossRefPubMedGoogle Scholar
  56. 56.
    Liu WJ, Xie SH, Liu YN et al (2012) Dipeptidyl peptidase IV inhibitor attenuates kidney injury in streptozotocin-induced diabetic rats. J Pharmacol Exp Ther 340:248–255CrossRefPubMedGoogle Scholar
  57. 57.
    Pyke C, Heller RS, Kirk RK et al (2014) GLP-1 receptor localization in monkey and human tissue: novel distribution revealed with extensively validated monoclonal antibody. Endocrinology 155:1280–1290CrossRefPubMedGoogle Scholar
  58. 58.
    Waser B, Blank A, Karamitopoulou E et al (2015) Glucagon-like-peptide-1 receptor expression in normal and diseased human thyroid and pancreas. Mod Pathol 28:391–402CrossRefPubMedGoogle Scholar
  59. 59.
    Wang QH, Chen K, Liu R et al (2010) Novel GLP-1 fusion chimera as potent long acting GLP-1 receptor agonist. PLoS One 5:9CrossRefGoogle Scholar
  60. 60.
    Levy OE, Jodka CM, Ren SS et al (2014) Novel exenatide analogs with peptidic albumin binding domains: potent anti-diabetic agents with extended duration of action. PLoS One 9:e87704PubMedCentralCrossRefPubMedGoogle Scholar
  61. 61.
    Glaesner W, Vick AM, Millican R et al (2010) Engineering and characterization of the long-acting glucagon-like peptide-1 analogue LY2189265, an Fc fusion protein. Diabetes Metab Res Rev 26:287–296CrossRefPubMedGoogle Scholar
  62. 62.
    Thurber GM, Schmidt MM, Wittrup KD (2008) Factors determining antibody distribution in tumors. Trends Pharmacol Sci 29:57–61PubMedCentralPubMedGoogle Scholar
  63. 63.
    Reynolds F, Kelly KA (2011) Techniques for molecular imaging probe design. Mol Imaging 10:407–419PubMedCentralPubMedGoogle Scholar
  64. 64.
    Mattes MJ, Griffiths G, Diril H et al (1994) Processing of antibody-radioisotope conjugates after binding to the surface of tumor cells. Cancer 73:787–793CrossRefPubMedGoogle Scholar
  65. 65.
    Wild D, Behe M, Wicki A et al (2006) [Lys(40) (Ahx-DTPA-In-111)NH2]exendin-4, a very promising ligand for glucagon-like peptide-1 (GLP-1) receptor targeting. J Nucl Med 47:2025–2033PubMedGoogle Scholar
  66. 66.
    Roed SN, Wismann P, Underwood CR et al (2014) Real-time trafficking and signaling of the glucagon-like peptide-1 receptor. Mol Cell Endocrinol 382:938–949CrossRefPubMedGoogle Scholar
  67. 67.
    Bavec A, Licar A (2009) Functional characterization of N-terminally GFP-tagged GLP-1 receptor. J Biomed BiotechnolGoogle Scholar
  68. 68.
    Schmidt MM, Wittrup KD (2009) A modeling analysis of the effects of molecular size and binding affinity on tumor targeting. Mol Cancer Ther 8:2861PubMedCentralCrossRefPubMedGoogle Scholar
  69. 69.
    Vegt E, Melis M, Eek A et al (2011) Renal uptake of different radiolabelled peptides is mediated by megalin: SPECT and biodistribution studies in megalin-deficient mice. Eur J Nucl Med Mol Imaging 38:623–632PubMedCentralCrossRefPubMedGoogle Scholar
  70. 70.
    Li ZB, Cai WB, Cao QZ et al (2007) 64Cu-labeled tetrameric and octameric RGD peptides for small-animal PET of tumor alpha(v)beta(3) integrin expression. J Nucl Med 48:1162–1171CrossRefPubMedGoogle Scholar
  71. 71.
    Cheng Z, Wu Y, Xiong ZM et al (2005) Near-infrared fluorescent RGD peptides for optical imaging of integrin alpha(v)beta 3 expression in living mice. Bioconjug Chem 16:1433–1441PubMedCentralCrossRefPubMedGoogle Scholar
  72. 72.
    Stern LA, Case BA, Hackel BJ (2013) Alternative non-antibody protein scaffolds for molecular imaging of cancer. Curr Opin Chem Eng 2Google Scholar
  73. 73.
    Moore SJ, Gephart MGH, Bergen JM et al (2013) Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma. Proc Natl Acad Sci U S A 110:14598–14603PubMedCentralCrossRefPubMedGoogle Scholar
  74. 74.
    Cilliers C, Liao J, Atangcho L, Thurber GM (2015) Residualization rates of near-infrared dyes for the rational design of molecular imaging agents. Mol Imaging BiolGoogle Scholar
  75. 75.
    Hahnenkamp A, Alsibai W, Bremer C, Holtke C (2014) Optimizing the bioavailability of small molecular optical imaging probes by conjugation to an albumin affinity tag. J Control Release 186:32–40CrossRefPubMedGoogle Scholar
  76. 76.
    Pollaro L, Raghunathan S, Morales-Sanfrutos J et al (2015) Bicyclic peptides conjugated to an albumin-binding tag diffuse efficiently into solid tumors. Mol Cancer Ther 14:151–161CrossRefPubMedGoogle Scholar
  77. 77.
    Geng SB, Cheung JK, Narasimhan C et al (2014) Improving monoclonal antibody selection and engineering using measurements of colloidal protein interactions. J Pharm Sci 103:3356–3363PubMedCentralCrossRefPubMedGoogle Scholar
  78. 78.
    Hotzel I, Theil FP, Bernstein LJ et al (2012) A strategy for risk mitigation of antibodies with fast clearance. MAbs 4:753–760PubMedCentralCrossRefPubMedGoogle Scholar
  79. 79.
    Tolmachev V, Tran TA, Rosik D et al (2012) Tumor targeting using affibody molecules: interplay of affinity, target expression level, and binding site composition. J Nucl Med 53:953–960CrossRefPubMedGoogle Scholar
  80. 80.
    Samkoe KS, Tichauer KM, Gunn JR et al (2014) Quantitative in vivo immunohistochemistry of epidermal growth factor receptor using a receptor concentration imaging approach. Cancer Res 74:7465–7474PubMedCentralCrossRefPubMedGoogle Scholar
  81. 81.
    Hampe CS, Wallen AR, Schlosser M et al (2005) Quantitative evaluation of a monoclonal antibody and its fragment as potential markers for pancreatic beta cell mass. Exp Clin Endocrinol Diabetes 113:381–387CrossRefPubMedGoogle Scholar
  82. 82.
    Kavishwar A, Moore A (2013) Sphingomyelin patches on pancreatic beta-cells are indicative of insulin secretory capacity. J Histochem Cytochem 61:910–919PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© World Molecular Imaging Society 2015

Authors and Affiliations

  1. 1.Department of Chemical EngineeringUniversity of MichiganAnn ArborUSA
  2. 2.Department of Biomedical EngineeringUniversity of MichiganAnn ArborUSA

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