Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1996 Aug 15;98(4):894–898. doi: 10.1172/JCI118871

Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells.

G Zeng 1, M J Quon 1
PMCID: PMC507502  PMID: 8770859

Abstract

Hypertension is associated with insulin-resistant states such as diabetes and obesity. Nitric oxide (NO) contributes to regulation of blood pressure. To gain insight into potential mechanisms linking hypertension with insulin resistance we directly measured and characterized NO production from human umbilical vein endothelial cells (HUVEC) in response to insulin using an amperometric NO-selective electrode. Insulin stimulation of HUVEC resulted in rapid, dose-dependent production of NO with a maximal response of approximately 100 nM NO (200,000 cells in 2 ml media; ED50 approximately 500 nM insulin). Although HUVEC have many more IGF-1 receptors than insulin receptors (approximately 400,000, and approximately 40,000 per cell respectively), a maximally stimulating dose of IGF-1 generated a smaller response than insulin (40 nM NO; ED50 approximately 100 nM IGF-1). Stimulation of HUVEC with PDGF did not result in measurable NO production. The effects of insulin and IGF-1 were completely blocked by inhibitors of either tyrosine kinase (genestein) or nitric oxide synthase (L-NAME). Wortmannin (an inhibitor of phosphatidylinositol 3-kinase [PI 3-kinase]) inhibited insulin-stimulated production of NO by approximately 50%. Since PI 3-kinase activity is required for insulin-stimulated glucose transport, our data suggest that NO is a novel effector of insulin signaling pathways that are also involved with glucose metabolism.

Full Text

The Full Text of this article is available as a PDF (174.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bar R. S., Boes M., Dake B. L., Booth B. A., Henley S. A., Sandra A. Insulin, insulin-like growth factors, and vascular endothelium. Am J Med. 1988 Nov 28;85(5A):59–70. doi: 10.1016/0002-9343(88)90398-1. [DOI] [PubMed] [Google Scholar]
  2. Bar R. S., Hoak J. C., Peacock M. L. Insulin receptors in human endothelial cells: identification and characterization. J Clin Endocrinol Metab. 1978 Sep;47(3):699–702. doi: 10.1210/jcem-47-3-699. [DOI] [PubMed] [Google Scholar]
  3. Baron A. D., Brechtel-Hook G., Johnson A., Hardin D. Skeletal muscle blood flow. A possible link between insulin resistance and blood pressure. Hypertension. 1993 Feb;21(2):129–135. doi: 10.1161/01.hyp.21.2.129. [DOI] [PubMed] [Google Scholar]
  4. Baron A. D., Brechtel G. Insulin differentially regulates systemic and skeletal muscle vascular resistance. Am J Physiol. 1993 Jul;265(1 Pt 1):E61–E67. doi: 10.1152/ajpendo.1993.265.1.E61. [DOI] [PubMed] [Google Scholar]
  5. Baron A. D. Hemodynamic actions of insulin. Am J Physiol. 1994 Aug;267(2 Pt 1):E187–E202. doi: 10.1152/ajpendo.1994.267.2.E187. [DOI] [PubMed] [Google Scholar]
  6. Baron A. D., Steinberg H. O., Chaker H., Leaming R., Johnson A., Brechtel G. Insulin-mediated skeletal muscle vasodilation contributes to both insulin sensitivity and responsiveness in lean humans. J Clin Invest. 1995 Aug;96(2):786–792. doi: 10.1172/JCI118124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Baron A. D., Steinberg H., Brechtel G., Johnson A. Skeletal muscle blood flow independently modulates insulin-mediated glucose uptake. Am J Physiol. 1994 Feb;266(2 Pt 1):E248–E253. doi: 10.1152/ajpendo.1994.266.2.E248. [DOI] [PubMed] [Google Scholar]
  8. Baron A. D. The coupling of glucose metabolism and perfusion in human skeletal muscle. The potential role of endothelium-derived nitric oxide. Diabetes. 1996 Jan;45 (Suppl 1):S105–S109. doi: 10.2337/diab.45.1.s105. [DOI] [PubMed] [Google Scholar]
  9. Bredt D. S., Snyder S. H. Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem. 1994;63:175–195. doi: 10.1146/annurev.bi.63.070194.001135. [DOI] [PubMed] [Google Scholar]
  10. Buchanan T. A., Meehan W. P., Jeng Y. Y., Yang D., Chan T. M., Nadler J. L., Scott S., Rude R. K., Hsueh W. A. Blood pressure lowering by pioglitazone. Evidence for a direct vascular effect. J Clin Invest. 1995 Jul;96(1):354–360. doi: 10.1172/JCI118041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cama A., Quon M. J., de la Luz Sierra M., Taylor S. I. Substitution of isoleucine for methionine at position 1153 in the beta-subunit of the human insulin receptor. A mutation that impairs receptor tyrosine kinase activity, receptor endocytosis, and insulin action. J Biol Chem. 1992 Apr 25;267(12):8383–8389. [PubMed] [Google Scholar]
  12. Claesson-Welsh L. Platelet-derived growth factor receptor signals. J Biol Chem. 1994 Dec 23;269(51):32023–32026. [PubMed] [Google Scholar]
  13. De Meyts P. The structural basis of insulin and insulin-like growth factor-I receptor binding and negative co-operativity, and its relevance to mitogenic versus metabolic signalling. Diabetologia. 1994 Sep;37 (Suppl 2):S135–S148. doi: 10.1007/BF00400837. [DOI] [PubMed] [Google Scholar]
  14. DeFronzo R. A., Ferrannini E. Insulin resistance. A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care. 1991 Mar;14(3):173–194. doi: 10.2337/diacare.14.3.173. [DOI] [PubMed] [Google Scholar]
  15. Ferrannini E., Buzzigoli G., Bonadonna R., Giorico M. A., Oleggini M., Graziadei L., Pedrinelli R., Brandi L., Bevilacqua S. Insulin resistance in essential hypertension. N Engl J Med. 1987 Aug 6;317(6):350–357. doi: 10.1056/NEJM198708063170605. [DOI] [PubMed] [Google Scholar]
  16. Furchgott R. F., Zawadzki J. V. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980 Nov 27;288(5789):373–376. doi: 10.1038/288373a0. [DOI] [PubMed] [Google Scholar]
  17. Giugliano D., De Rosa N., Di Maro G., Marfella R., Acampora R., Buoninconti R., D'Onofrio F. Metformin improves glucose, lipid metabolism, and reduces blood pressure in hypertensive, obese women. Diabetes Care. 1993 Oct;16(10):1387–1390. doi: 10.2337/diacare.16.10.1387. [DOI] [PubMed] [Google Scholar]
  18. Huang P. L., Huang Z., Mashimo H., Bloch K. D., Moskowitz M. A., Bevan J. A., Fishman M. C. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature. 1995 Sep 21;377(6546):239–242. doi: 10.1038/377239a0. [DOI] [PubMed] [Google Scholar]
  19. Ignarro L. J., Buga G. M., Wood K. S., Byrns R. E., Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9265–9269. doi: 10.1073/pnas.84.24.9265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Isakoff S. J., Taha C., Rose E., Marcusohn J., Klip A., Skolnik E. Y. The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10247–10251. doi: 10.1073/pnas.92.22.10247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. King G. L., Johnson S. M. Receptor-mediated transport of insulin across endothelial cells. Science. 1985 Mar 29;227(4694):1583–1586. doi: 10.1126/science.3883490. [DOI] [PubMed] [Google Scholar]
  22. Laakso M., Edelman S. V., Brechtel G., Baron A. D. Decreased effect of insulin to stimulate skeletal muscle blood flow in obese man. A novel mechanism for insulin resistance. J Clin Invest. 1990 Jun;85(6):1844–1852. doi: 10.1172/JCI114644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Laakso M., Edelman S. V., Brechtel G., Baron A. D. Impaired insulin-mediated skeletal muscle blood flow in patients with NIDDM. Diabetes. 1992 Sep;41(9):1076–1083. doi: 10.2337/diab.41.9.1076. [DOI] [PubMed] [Google Scholar]
  24. Lee M. K., Miles P. D., Khoursheed M., Gao K. M., Moossa A. R., Olefsky J. M. Metabolic effects of troglitazone on fructose-induced insulin resistance in the rat. Diabetes. 1994 Dec;43(12):1435–1439. doi: 10.2337/diab.43.12.1435. [DOI] [PubMed] [Google Scholar]
  25. Ogihara T., Rakugi H., Ikegami H., Mikami H., Masuo K. Enhancement of insulin sensitivity by troglitazone lowers blood pressure in diabetic hypertensives. Am J Hypertens. 1995 Mar;8(3):316–320. doi: 10.1016/0895-7061(95)96214-5. [DOI] [PubMed] [Google Scholar]
  26. Palmer R. M., Ferrige A. G., Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987 Jun 11;327(6122):524–526. doi: 10.1038/327524a0. [DOI] [PubMed] [Google Scholar]
  27. Quon M. J., Chen H., Ing B. L., Liu M. L., Zarnowski M. J., Yonezawa K., Kasuga M., Cushman S. W., Taylor S. I. Roles of 1-phosphatidylinositol 3-kinase and ras in regulating translocation of GLUT4 in transfected rat adipose cells. Mol Cell Biol. 1995 Oct;15(10):5403–5411. doi: 10.1128/mcb.15.10.5403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Quon M. J., Guerre-Millo M., Zarnowski M. J., Butte A. J., Em M., Cushman S. W., Taylor S. I. Tyrosine kinase-deficient mutant human insulin receptors (Met1153-->Ile) overexpressed in transfected rat adipose cells fail to mediate translocation of epitope-tagged GLUT4. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5587–5591. doi: 10.1073/pnas.91.12.5587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Reaven G. M., Lithell H., Landsberg L. Hypertension and associated metabolic abnormalities--the role of insulin resistance and the sympathoadrenal system. N Engl J Med. 1996 Feb 8;334(6):374–381. doi: 10.1056/NEJM199602083340607. [DOI] [PubMed] [Google Scholar]
  30. Reaven G. M. Pathophysiology of insulin resistance in human disease. Physiol Rev. 1995 Jul;75(3):473–486. doi: 10.1152/physrev.1995.75.3.473. [DOI] [PubMed] [Google Scholar]
  31. Steinberg H. O., Brechtel G., Johnson A., Fineberg N., Baron A. D. Insulin-mediated skeletal muscle vasodilation is nitric oxide dependent. A novel action of insulin to increase nitric oxide release. J Clin Invest. 1994 Sep;94(3):1172–1179. doi: 10.1172/JCI117433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tsukahara H., Gordienko D. V., Tonshoff B., Gelato M. C., Goligorsky M. S. Direct demonstration of insulin-like growth factor-I-induced nitric oxide production by endothelial cells. Kidney Int. 1994 Feb;45(2):598–604. doi: 10.1038/ki.1994.78. [DOI] [PubMed] [Google Scholar]
  33. Valius M., Kazlauskas A. Phospholipase C-gamma 1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor's mitogenic signal. Cell. 1993 Apr 23;73(2):321–334. doi: 10.1016/0092-8674(93)90232-f. [DOI] [PubMed] [Google Scholar]
  34. Wennström S., Hawkins P., Cooke F., Hara K., Yonezawa K., Kasuga M., Jackson T., Claesson-Welsh L., Stephens L. Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling. Curr Biol. 1994 May 1;4(5):385–393. doi: 10.1016/s0960-9822(00)00087-7. [DOI] [PubMed] [Google Scholar]
  35. Wennström S., Siegbahn A., Yokote K., Arvidsson A. K., Heldin C. H., Mori S., Claesson-Welsh L. Membrane ruffling and chemotaxis transduced by the PDGF beta-receptor require the binding site for phosphatidylinositol 3' kinase. Oncogene. 1994 Feb;9(2):651–660. [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES