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. 1997 Jan 1;99(1):14–18. doi: 10.1172/JCI119125

Human blood-brain barrier leptin receptor. Binding and endocytosis in isolated human brain microvessels.

P L Golden 1, T J Maccagnan 1, W M Pardridge 1
PMCID: PMC507761  PMID: 9011568

Abstract

The peripheral production of leptin by adipose tissue and its putative effect as a signal of satiety in the central nervous system suggest that leptin gains access to the regions of the brain regulating energy balance by crossing the brain capillary endothelium, which constitutes the blood-brain barrier in vivo. The present experiments characterize the binding and internalization of mouse recombinant leptin in isolated human brain capillaries, an in vitro model of the human blood-brain barrier. Incubation of 125I-leptin with isolated human brain capillaries resulted in temperature-dependent binding: at 37 degrees C, approximately 65% of radiolabeled leptin was bound per milligram of capillary protein. Two-thirds of the bound radioactivity was resistant to removal by acid wash, demonstrating endocytosis of 125I-leptin into capillary cells. At 4 degrees C, binding to isolated capillaries was reduced to approximately 23%/mg of protein, the majority of which was acid wash resistant. Binding of 125I-leptin to brain capillary endothelial plasma membranes was saturable, described by a two-site binding model with a high-affinity dissociation constant of 5.1+/-2.8 nM and maximal binding capacity of 0.34+/-0.16 pmol/mg of membrane protein. Addition of porcine insulin or insulin-like growth factor at a final concentration of 100 nM had a negligible effect on leptin binding. These results provide evidence for a leptin receptor that mediates saturable, specific, temperature-dependent binding and endocytosis of leptin at the human blood-brain barrier.

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Selected References

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  1. Banks W. A., Kastin A. J., Huang W., Jaspan J. B., Maness L. M. Leptin enters the brain by a saturable system independent of insulin. Peptides. 1996;17(2):305–311. doi: 10.1016/0196-9781(96)00025-3. [DOI] [PubMed] [Google Scholar]
  2. Becker D. J., Ongemba L. N., Brichard V., Henquin J. C., Brichard S. M. Diet- and diabetes-induced changes of ob gene expression in rat adipose tissue. FEBS Lett. 1995 Sep 11;371(3):324–328. doi: 10.1016/0014-5793(95)00943-4. [DOI] [PubMed] [Google Scholar]
  3. Bickel U., Kang Y. S., Yoshikawa T., Pardridge W. M. In vivo demonstration of subcellular localization of anti-transferrin receptor monoclonal antibody-colloidal gold conjugate in brain capillary endothelium. J Histochem Cytochem. 1994 Nov;42(11):1493–1497. doi: 10.1177/42.11.7930531. [DOI] [PubMed] [Google Scholar]
  4. Bolton A. E., Hunter W. M. The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem J. 1973 Jul;133(3):529–539. doi: 10.1042/bj1330529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Campfield L. A., Smith F. J., Guisez Y., Devos R., Burn P. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science. 1995 Jul 28;269(5223):546–549. doi: 10.1126/science.7624778. [DOI] [PubMed] [Google Scholar]
  6. Carpentier J. L., Fehlmann M., Van Obberghen E., Gorden P., Orci L. Redistribution of 125I-insulin on the surface of rat hepatocytes as a function of dissociation time. Diabetes. 1985 Oct;34(10):1002–1007. doi: 10.2337/diab.34.10.1002. [DOI] [PubMed] [Google Scholar]
  7. Chen H., Charlat O., Tartaglia L. A., Woolf E. A., Weng X., Ellis S. J., Lakey N. D., Culpepper J., Moore K. J., Breitbart R. E. Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell. 1996 Feb 9;84(3):491–495. doi: 10.1016/s0092-8674(00)81294-5. [DOI] [PubMed] [Google Scholar]
  8. Considine R. V., Sinha M. K., Heiman M. L., Kriauciunas A., Stephens T. W., Nyce M. R., Ohannesian J. P., Marco C. C., McKee L. J., Bauer T. L. Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med. 1996 Feb 1;334(5):292–295. doi: 10.1056/NEJM199602013340503. [DOI] [PubMed] [Google Scholar]
  9. Devos R., Richards J. G., Campfield L. A., Tartaglia L. A., Guisez Y., van der Heyden J., Travernier J., Plaetinck G., Burn P. OB protein binds specifically to the choroid plexus of mice and rats. Proc Natl Acad Sci U S A. 1996 May 28;93(11):5668–5673. doi: 10.1073/pnas.93.11.5668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duffy K. R., Pardridge W. M. Blood-brain barrier transcytosis of insulin in developing rabbits. Brain Res. 1987 Sep 8;420(1):32–38. doi: 10.1016/0006-8993(87)90236-8. [DOI] [PubMed] [Google Scholar]
  11. Duffy K. R., Pardridge W. M., Rosenfeld R. G. Human blood-brain barrier insulin-like growth factor receptor. Metabolism. 1988 Feb;37(2):136–140. doi: 10.1016/s0026-0495(98)90007-5. [DOI] [PubMed] [Google Scholar]
  12. Feldman H. A. Mathematical theory of complex ligand-binding systems of equilibrium: some methods for parameter fitting. Anal Biochem. 1972 Aug;48(2):317–338. doi: 10.1016/0003-2697(72)90084-x. [DOI] [PubMed] [Google Scholar]
  13. Fishman J. B., Rubin J. B., Handrahan J. V., Connor J. R., Fine R. E. Receptor-mediated transcytosis of transferrin across the blood-brain barrier. J Neurosci Res. 1987;18(2):299–304. doi: 10.1002/jnr.490180206. [DOI] [PubMed] [Google Scholar]
  14. Frank H. J., Pardridge W. M., Morris W. L., Rosenfeld R. G., Choi T. B. Binding and internalization of insulin and insulin-like growth factors by isolated brain microvessels. Diabetes. 1986 Jun;35(6):654–661. doi: 10.2337/diab.35.6.654. [DOI] [PubMed] [Google Scholar]
  15. Frederich R. C., Löllmann B., Hamann A., Napolitano-Rosen A., Kahn B. B., Lowell B. B., Flier J. S. Expression of ob mRNA and its encoded protein in rodents. Impact of nutrition and obesity. J Clin Invest. 1995 Sep;96(3):1658–1663. doi: 10.1172/JCI118206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Halaas J. L., Gajiwala K. S., Maffei M., Cohen S. L., Chait B. T., Rabinowitz D., Lallone R. L., Burley S. K., Friedman J. M. Weight-reducing effects of the plasma protein encoded by the obese gene. Science. 1995 Jul 28;269(5223):543–546. doi: 10.1126/science.7624777. [DOI] [PubMed] [Google Scholar]
  17. Lee G. H., Proenca R., Montez J. M., Carroll K. M., Darvishzadeh J. G., Lee J. I., Friedman J. M. Abnormal splicing of the leptin receptor in diabetic mice. Nature. 1996 Feb 15;379(6566):632–635. doi: 10.1038/379632a0. [DOI] [PubMed] [Google Scholar]
  18. Leroy P., Dessolin S., Villageois P., Moon B. C., Friedman J. M., Ailhaud G., Dani C. Expression of ob gene in adipose cells. Regulation by insulin. J Biol Chem. 1996 Feb 2;271(5):2365–2368. doi: 10.1074/jbc.271.5.2365. [DOI] [PubMed] [Google Scholar]
  19. Lidinsky W. A., Drewes L. R. Characterization of the blood-brain barrier: protein composition of the capillary endothelial cell membrane. J Neurochem. 1983 Nov;41(5):1341–1348. doi: 10.1111/j.1471-4159.1983.tb00831.x. [DOI] [PubMed] [Google Scholar]
  20. Lynn R. B., Cao G. Y., Considine R. V., Hyde T. M., Caro J. F. Autoradiographic localization of leptin binding in the choroid plexus of ob/ob and db/db mice. Biochem Biophys Res Commun. 1996 Feb 27;219(3):884–889. doi: 10.1006/bbrc.1996.0328. [DOI] [PubMed] [Google Scholar]
  21. Malik K. F., Young W. S., 3rd Localization of binding sites in the central nervous system for leptin (OB protein) in normal, obese (ob/ob), and diabetic (db/db) C57BL/6J mice. Endocrinology. 1996 Apr;137(4):1497–1500. doi: 10.1210/endo.137.4.8625929. [DOI] [PubMed] [Google Scholar]
  22. Masuzaki H., Ogawa Y., Isse N., Satoh N., Okazaki T., Shigemoto M., Mori K., Tamura N., Hosoda K., Yoshimasa Y. Human obese gene expression. Adipocyte-specific expression and regional differences in the adipose tissue. Diabetes. 1995 Jul;44(7):855–858. doi: 10.2337/diab.44.7.855. [DOI] [PubMed] [Google Scholar]
  23. McGregor G. P., Desaga J. F., Ehlenz K., Fischer A., Heese F., Hegele A., Lammer C., Peiser C., Lang R. E. Radiommunological measurement of leptin in plasma of obese and diabetic human subjects. Endocrinology. 1996 Apr;137(4):1501–1504. doi: 10.1210/endo.137.4.8625930. [DOI] [PubMed] [Google Scholar]
  24. Moore M. S., Mahaffey D. T., Brodsky F. M., Anderson R. G. Assembly of clathrin-coated pits onto purified plasma membranes. Science. 1987 May 1;236(4801):558–563. doi: 10.1126/science.2883727. [DOI] [PubMed] [Google Scholar]
  25. Olefsky J. M., Kao M. Surface binding and rates of internalization of 125I-insulin in adipocytes and IM-9 lymphocytes. J Biol Chem. 1982 Aug 10;257(15):8667–8673. [PubMed] [Google Scholar]
  26. Pardridge W. M., Eisenberg J., Yamada T. Rapid sequestration and degradation of somatostatin analogues by isolated brain microvessels. J Neurochem. 1985 Apr;44(4):1178–1184. doi: 10.1111/j.1471-4159.1985.tb08741.x. [DOI] [PubMed] [Google Scholar]
  27. Pardridge W. M., Eisenberg J., Yang J. Human blood-brain barrier insulin receptor. J Neurochem. 1985 Jun;44(6):1771–1778. doi: 10.1111/j.1471-4159.1985.tb07167.x. [DOI] [PubMed] [Google Scholar]
  28. Pardridge W. M., Eisenberg J., Yang J. Human blood-brain barrier transferrin receptor. Metabolism. 1987 Sep;36(9):892–895. doi: 10.1016/0026-0495(87)90099-0. [DOI] [PubMed] [Google Scholar]
  29. Pardridge W. M. Receptor-mediated peptide transport through the blood-brain barrier. Endocr Rev. 1986 Aug;7(3):314–330. doi: 10.1210/edrv-7-3-314. [DOI] [PubMed] [Google Scholar]
  30. Pelleymounter M. A., Cullen M. J., Baker M. B., Hecht R., Winters D., Boone T., Collins F. Effects of the obese gene product on body weight regulation in ob/ob mice. Science. 1995 Jul 28;269(5223):540–543. doi: 10.1126/science.7624776. [DOI] [PubMed] [Google Scholar]
  31. Reinhardt R. R., Bondy C. A. Insulin-like growth factors cross the blood-brain barrier. Endocrinology. 1994 Nov;135(5):1753–1761. doi: 10.1210/endo.135.5.7525251. [DOI] [PubMed] [Google Scholar]
  32. Saladin R., De Vos P., Guerre-Millo M., Leturque A., Girard J., Staels B., Auwerx J. Transient increase in obese gene expression after food intake or insulin administration. Nature. 1995 Oct 12;377(6549):527–529. doi: 10.1038/377527a0. [DOI] [PubMed] [Google Scholar]
  33. Schwartz M. W., Baskin D. G., Bukowski T. R., Kuijper J. L., Foster D., Lasser G., Prunkard D. E., Porte D., Jr, Woods S. C., Seeley R. J. Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice. Diabetes. 1996 Apr;45(4):531–535. doi: 10.2337/diab.45.4.531. [DOI] [PubMed] [Google Scholar]
  34. Slieker L. J., Sloop K. W., Surface P. L., Kriauciunas A., LaQuier F., Manetta J., Bue-Valleskey J., Stephens T. W. Regulation of expression of ob mRNA and protein by glucocorticoids and cAMP. J Biol Chem. 1996 Mar 8;271(10):5301–5304. doi: 10.1074/jbc.271.10.5301. [DOI] [PubMed] [Google Scholar]
  35. Stephens T. W., Basinski M., Bristow P. K., Bue-Valleskey J. M., Burgett S. G., Craft L., Hale J., Hoffmann J., Hsiung H. M., Kriauciunas A. The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature. 1995 Oct 12;377(6549):530–532. doi: 10.1038/377530a0. [DOI] [PubMed] [Google Scholar]
  36. Tartaglia L. A., Dembski M., Weng X., Deng N., Culpepper J., Devos R., Richards G. J., Campfield L. A., Clark F. T., Deeds J. Identification and expression cloning of a leptin receptor, OB-R. Cell. 1995 Dec 29;83(7):1263–1271. doi: 10.1016/0092-8674(95)90151-5. [DOI] [PubMed] [Google Scholar]
  37. Weigle D. S., Bukowski T. R., Foster D. C., Holderman S., Kramer J. M., Lasser G., Lofton-Day C. E., Prunkard D. E., Raymond C., Kuijper J. L. Recombinant ob protein reduces feeding and body weight in the ob/ob mouse. J Clin Invest. 1995 Oct;96(4):2065–2070. doi: 10.1172/JCI118254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zhang Y., Proenca R., Maffei M., Barone M., Leopold L., Friedman J. M. Positional cloning of the mouse obese gene and its human homologue. Nature. 1994 Dec 1;372(6505):425–432. doi: 10.1038/372425a0. [DOI] [PubMed] [Google Scholar]

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