Measuring Phospholipase D Activity in Insulin-Secreting Pancreatic β-Cells and Insulin-Responsive Muscle Cells and Adipocytes

  • Rosanna Cazzolli
  • Ping Huang
  • Shuzhi Teng
  • William E Hughes
Part of the Methods in Molecular Biology book series (MIMB, volume 462)


Phospholipase D (PLD) is an enzyme producing phosphatidic acid and choline through hydrolysis of phosphatidylcholine. The enzyme has been identified as a member of a variety of signal transduction cascades and as a key regulator of numerous intracellular vesicle trafficking processes. A role for PLD in regulating glucose homeostasis is emerging as the enzyme has recently been identified in events regulating exocytosis of insulin from pancreatic β-cells and also in insulin-stimulated glucose uptake through controlling GLUT4 vesicle exocytosis in muscle and adipose tissue. We present methodologies for assessing cellular PLD activity in secretagogue-stimulated insulin-secreting pancreatic β-cells and also insulin-stimulated adipocyte and muscle cells, two of the principal insulin-responsive cell types controlling blood glucose levels.


Phospholipase D insulin GLUT4 adipocyte 3T3-L1 myotube L6 MIN6 pancreatic β-cell 


  1. 1.
    Hanahan DJ, Chaikoff IL. The phosphorous-containing lipides of the carrot. J Biol Chem 1947;168:233–40.PubMedGoogle Scholar
  2. 2.
    Exton JH. Phospholipase D-structure, regulation and function. Rev Physiol Biochem Pharmacol 2002;144:1–94.PubMedCrossRefGoogle Scholar
  3. 3.
    Jenkins GM, Frohman, MA. Phospholipase D: A lipid centric review. Cell Mol Life Sci 2005;62:2305–16.PubMedCrossRefGoogle Scholar
  4. 4.
    Chen YG, Siddhanta A, Austin CD, Hammond SM, Sung TC, Frohman MA, Morris AJ, Shields D. Phospholipase D stimulates release of nascent secretory vesicles from the trans-Golgi network. J Cell Biol 1997;138:495–504.PubMedCrossRefGoogle Scholar
  5. 5.
    Shen Y, Xu L, Foster DA. Role for phospholipase D in receptor-mediated endocytosis. Mol Cell Biol 2001;21:595–602.PubMedCrossRefGoogle Scholar
  6. 6.
    Humeau Y, Vitale N, Chasserot-Golaz S, Dupont JL, Du G, Frohman MA, Bader MF, Poulain B. A role for phospholipase D1 in neurotransmitter release. Proc Natl Acad Sci USA 2001;98:15300–5.PubMedCrossRefGoogle Scholar
  7. 7.
    Hughes WE, Elgundi Z, Huang P, Frohman MA, Biden TJ. Phospholipase D1 regulates secretagogue-stimulated insulin release in pancreatic beta-cells. J Biol Chem 2004;279:27534–41.PubMedCrossRefGoogle Scholar
  8. 8.
    Vitale N, Caumont AS, Chasserot-Golaz S, Du G, Wu S, Sciorra VA, Morris AJ, Frohman MA, Bader MF. Phospholipase D1: A key factor for the exocytotic machinery in neuroendocrine cells. EMBO J 2001;20:2424–34.PubMedCrossRefGoogle Scholar
  9. 9.
    Emoto M, Klarlund JK, Waters SB, Hu V, Buxton JM, Chawla A, Czech MP. A role for phospholipase D in GLUT4 glucose transporter translocation. J Biol Chem 2001;275:7144–51.CrossRefGoogle Scholar
  10. 10.
    Huang P, Altshuller YM, Hou JC, Pessin JE, Frohman MA. Insulin-stimulated plasma membrane fusion of Glut4 glucose transporter-containing vesicles is regulated by phospholipase D1. Mol Biol Cell 2005;16:2614–23.PubMedCrossRefGoogle Scholar
  11. 11.
    Straub SG, Sharp GW. Glucose-stimulated signaling pathways in biphasic insulin secretion. Diabetes Metab Res Rev 2002;18:451–63.PubMedCrossRefGoogle Scholar
  12. 12.
    Gilon P, Henquin JC. Mechanisms and physiological significance of the cholinergic control of pancreatic beta-cell function. Endocr Rev 2001;22:565–604.PubMedCrossRefGoogle Scholar
  13. 13.
    Bratanova-Tochkova TK, Cheng H, Daniel S, Gunawardana S, Liu YJ, Mulvaney-Musa J, Schermerhorn T, Straub SG, Yajima H, Sharp GW. Triggering and augmentation mechanisms, granule pools and biphasic insulin secretion. Diabetes 2002; 51(Suppl 1):S83–S90.PubMedCrossRefGoogle Scholar
  14. 14.
    Taniguchi CM, Emanuelli B, Kahn CR. Critical nodes in signalling pathways: Insights into insulin action. Nat Rev Mol Cell Biol 2006;7:85–96.PubMedCrossRefGoogle Scholar
  15. 15.
    Bryant NJ, Govers R, James DE. Regulated transport of the glucose transporter GLUT4. Nat Rev Mol Cell Biol 2002;3:267–77.PubMedCrossRefGoogle Scholar
  16. 16.
    Morris AJ, Frohman MA, Engebrecht J. Measurement of phospholipase D activity. Anal Biochem 1997;252:1–9.PubMedCrossRefGoogle Scholar
  17. 17.
    Bogan JS, McKee AE, Lodish HF. Insulin-responsive compartments containing GLUT4 in 3T3-L1 and CHO cells: Regulation by amino acid concentrations. Mol Cell Biol 2001;21:4785–806.PubMedCrossRefGoogle Scholar
  18. 18.
    Hughes WE, Larijani B, Parker PJ. Detecting protein-phospholipid interactions. Epidermal growth factor-induced activation of phospholipase D1b in situ. J Biol Chem 2002;277:22974–9.PubMedCrossRefGoogle Scholar
  19. 19.
    Seiler AE, Henderson A, Rubin R. Ethanol inhibits insulin receptor tyrosine kinase. Alcohol Clin Exp Res 2000;24:1869–72.PubMedCrossRefGoogle Scholar
  20. 20.
    Xu J, Yeon JE, Chang H, Tison G, Chen GJ, Wands J, de la Monte S. (2003) Ethanol impairs insulin-stimulated neuronal survival in the developing brain: Role of PTEN phosphatase. J Biol Chem 278:26929–37.PubMedCrossRefGoogle Scholar
  21. 21.
    Miyazaki J, Araki K, Yamato E, Ikegami H, Asano T, Shibasaki Y, Oka Y, Yamamura K. Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: Special reference to expression of glucose transporter isoforms. Endocrinology 1990;127:126–32.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Rosanna Cazzolli
  • Ping Huang
  • Shuzhi Teng
  • William E Hughes
    • 1
  1. 1.Phospholipid Biology Group, Department of MedicineGarvan Institute of Medical Research, University of New South WalesSydneyAustralia

Personalised recommendations