title: Characterization of P4 ATPase Phospholipid Translocases (Flippases) in Human and Rat Pancreatic Beta Cells: THEIR GENE SILENCING INHIBITS INSULIN SECRETION.
abstract: The negative charge of phosphatidylserine in lipid bilayers of secretory vesicles and plasma membranes couples the domains of positively charged amino acids of secretory vesicle SNARE proteins with similar domains of plasma membrane SNARE proteins enhancing fusion of the two membranes to promote exocytosis of the vesicle contents of secretory cells. Our recent study of insulin secretory granules (ISG) (MacDonald, M. J., Ade, L., Ntambi, J. M., Ansari, I. H., and Stoker, S. W. (2015) Characterization of phospholipids in insulin secretory granules in pancreatic beta cells and their changes with glucose stimulation. J. Biol. Chem. 290, 11075-11092) suggested that phosphatidylserine and other phospholipids, such as phosphatidylethanolamine, in ISG could play important roles in docking and fusion of ISG to the plasma membrane in the pancreatic beta cell during insulin exocytosis. P4 ATPase flippases translocate primarily phosphatidylserine and, to a lesser extent, phosphatidylethanolamine across the lipid bilayers of intracellular vesicles and plasma membranes to the cytosolic leaflets of these membranes. CDC50A is a protein that forms a heterodimer with P4 ATPases to enhance their translocase catalytic activity. We found that the predominant P4 ATPases in pure pancreatic beta cells and human and rat pancreatic islets were ATP8B1, ATP8B2, and ATP9A. ATP8B1 and CDC50A were highly concentrated in ISG. ATP9A was concentrated in plasma membrane. Gene silencing of individual P4 ATPases and CDC50A inhibited glucose-stimulated insulin release in pure beta cells and in human pancreatic islets. This is the first characterization of P4 ATPases in beta cells. The results support roles for P4 ATPases in translocating phosphatidylserine to the cytosolic leaflets of ISG and the plasma membrane to facilitate the docking and fusion of ISG to the plasma membrane during insulin exocytosis.
authors.affiliation: From the Childrens Diabetes Center, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706 and.
authors.forename: Israr-ul H
authors.initials: IU
authors.lastname: Ansari
authors.affiliation: From the Childrens Diabetes Center, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706 and.
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Longacre
authors.affiliation: the Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, 1105 BK Amsterdam, The Netherlands.
authors.forename: Coen C
authors.initials: CC
authors.lastname: Paulusma
authors.affiliation: From the Childrens Diabetes Center, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706 and.
authors.forename: Scott W
authors.initials: SW
authors.lastname: Stoker
authors.affiliation: From the Childrens Diabetes Center, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706 and.
authors.forename: Mindy A
authors.initials: MA
authors.lastname: Kendrick
authors.affiliation: From the Childrens Diabetes Center, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706 and mjmacdon@wisc.edu.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: MacDonald
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D020868:Gene Silencing; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D010718:Phosphatidylserines / Q000235:genetics / Q000378:metabolism; D049294:Phospholipid Transfer Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats
chemical_list: D007328:Insulin; D008565:Membrane Proteins; D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; C526978:TMEM30a protein, human; C545244:Tmem30a protein, rat; D000251:Adenosine Triphosphatases
keywords: P4 ATPases; beta cell (B-cell); gene silencing; human pancreatic islets; insulin exocytosis; insulin secretion; insulin secretory vesicles; phosphatidylserine; phospholipid; phospholipid translocases