title: Subplasmalemmal Ca(2+) measurements in mouse pancreatic beta cells support the existence of an amplifying effect of glucose on insulin secretion.
abstract: OBJECTIVE
  Glucose-induced insulin secretion is attributed to a rise of beta cell cytosolic free [Ca(2+)] ([Ca(2+)](c)) (triggering pathway) and amplification of the action of Ca(2+). This concept of amplification rests on observations that glucose can increase Ca(2+)-induced insulin secretion without further elevating an imposed already high [Ca(2+)](c). However, it remains possible that this amplification results from an increase in [Ca(2+)] just under the plasma membrane ([Ca(2+)](SM)), which escaped detection by previous measurements of global [Ca(2+)](c). This was the hypothesis that we tested here by measuring [Ca(2+)](SM).
  
  
  METHODS
  The genetically encoded Ca(2+) indicators D3-cpv (untargeted) and LynD3-cpv (targeted to plasma membrane) were expressed in clusters of mouse beta cells. LynD3-cpv was also expressed in beta cells within intact islets. [Ca(2+)](SM) changes were monitored using total internal reflection fluorescence microscopy. Insulin secretion was measured in parallel.
  
  
  RESULTS
  Beta cells expressing D3cpv or LynD3cpv displayed normal [Ca(2+)] changes and insulin secretion in response to glucose. Distinct [Ca(2+)](SM) fluctuations were detected during repetitive variations of KCl between 30 and 32-35 mmol/l, attesting to the adequate sensitivity of our system. When the amplifying pathway was evaluated (high KCl + diazoxide), increasing glucose from 3 to 15 mmol/l consistently lowered [Ca(2+)](SM) while stimulating insulin secretion approximately two fold. Blocking Ca(2+) uptake by the endoplasmic reticulum largely attenuated the [Ca(2+)](SM) decrease produced by high glucose but did not unmask localised [Ca(2+)](SM) increases.
  
  
  CONCLUSIONS
  Glucose can increase Ca(2+)-induced insulin secretion without causing further elevation of beta cell [Ca(2+)](SM). The phenomenon is therefore a true amplification of the triggering action of Ca(2+).
authors.affiliation: Unit of Endocrinology and Metabolism, University of Louvain Faculty of Medicine, UCL 55.30, Brussels, Belgium.
authors.forename: M A
authors.initials: MA
authors.lastname: Ravier
authors.forename: R
authors.initials: R
authors.lastname: Cheng-Xue
authors.forename: A E
authors.initials: AE
authors.lastname: Palmer
authors.forename: J C
authors.initials: JC
authors.lastname: Henquin
authors.forename: P
authors.initials: P
authors.lastname: Gilon
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D005947:Glucose / Q000494:pharmacology*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence
chemical_list: D007328:Insulin; D005947:Glucose; D002118:Calcium
references.citation: Methods. 2008 Nov;46(3):152-9
references.pmid: 18848629
references.citation: Am J Physiol Endocrinol Metab. 2006 Jan;290(1):E143-E148
references.pmid: 16144817
references.citation: EMBO J. 1995 Jan 3;14(1):50-7
references.pmid: 7828595
references.citation: J Biol Chem. 2003 Jun 20;278(25):22956-63
references.pmid: 12682074
references.citation: Cell Calcium. 2006 Nov-Dec;40(5-6):539-51
references.pmid: 17030367
references.citation: Proc Natl Acad Sci U S A. 2005 Jul 26;102(30):10700-5
references.pmid: 16027365
references.citation: Diabetes. 2004 Feb;53(2):393-400
references.pmid: 14747290
references.citation: J Cell Biol. 1997 Jul 14;138(1):55-64
references.pmid: 9214381
references.citation: Methods. 2008 Nov;46(3):233-8
references.pmid: 18854212
references.citation: J Biol Chem. 2002 Oct 4;277(40):37702-10
references.pmid: 12149258
references.citation: Eur J Clin Invest. 2003 Sep;33(9):742-50
references.pmid: 12925032
references.citation: Diabetes. 2006 Dec;55(12):3470-7
references.pmid: 17130494
references.citation: Nature. 1989 Nov 30;342(6249):550-3
references.pmid: 2479839
references.citation: Nat Methods. 2008 Sep;5(9):797-804
references.pmid: 19160514
references.citation: J Biol Chem. 2004 Jun 4;279(23):24685-91
references.pmid: 15073181
references.citation: Endocrinology. 1999 May;140(5):2252-7
references.pmid: 10218978
references.citation: Cell Calcium. 2008 Dec;44(6):533-44
references.pmid: 18452988
references.citation: Endocrinology. 2009 Jan;150(1):33-45
references.pmid: 18787024
references.citation: Proc Natl Acad Sci U S A. 2009 Apr 14;106(15):6404-9
references.pmid: 19332787
references.citation: J Clin Invest. 1996 Jun 1;97(11):2417-25
references.pmid: 8647933
references.citation: Proc Natl Acad Sci U S A. 2009 Nov 17;106(46):19358-62
references.pmid: 19906989
references.citation: Am J Physiol Endocrinol Metab. 2009 Apr;296(4):E690-701
references.pmid: 19141690
references.citation: Nat Neurosci. 2003 Aug;6(8):846-53
references.pmid: 12845327
references.citation: Diabetes. 1998 May;47(5):758-63
references.pmid: 9588447
references.citation: Diabetes. 2005 Jul;54(7):2132-42
references.pmid: 15983215
references.citation: J Physiol. 1997 Sep 1;503 ( Pt 2):399-412
references.pmid: 9306281
references.citation: Diabetologia. 2003 Aug;46(8):1029-45
references.pmid: 12879249
references.citation: Science. 2002 May 3;296(5569):913-6
references.pmid: 11988576
references.citation: Naunyn Schmiedebergs Arch Pharmacol. 1988 Oct;338(4):459-62
references.pmid: 3149719
references.citation: Endocrine. 2000 Dec;13(3):251-62
references.pmid: 11216635
references.citation: J Clin Invest. 1992 Apr;89(4):1288-95
references.pmid: 1556189
references.citation: Nat Cell Biol. 2003 Apr;5(4):330-5
references.pmid: 12640462
references.citation: Cell Metab. 2009 Dec;10(6):455-65
references.pmid: 19945403
references.citation: J Biol Chem. 1992 Sep 5;267(25):18110-7
references.pmid: 1517243
references.citation: J Biol Chem. 1999 May 7;274(19):13281-91
references.pmid: 10224088
references.citation: J Physiol. 2000 May 15;525 Pt 1:159-67
references.pmid: 10811734
references.citation: Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):760-5
references.pmid: 9892707
references.citation: J Gen Physiol. 2005 Sep;126(3):179-92
references.pmid: 16103278
references.citation: Am J Physiol Endocrinol Metab. 2008 Dec;295(6):E1298-306
references.pmid: 18940941
references.citation: J Physiol. 2008 Jul 15;586(14):3313-24
references.pmid: 18511483
references.citation: Diabetes Metab Res Rev. 2002 Nov-Dec;18(6):451-63
references.pmid: 12469359
references.citation: J Biol Chem. 1992 Oct 15;267(29):20713-20
references.pmid: 1400388
references.citation: Proc Natl Acad Sci U S A. 2007 Jan 30;104(5):1697-702
references.pmid: 17242349
references.citation: Biophys J. 2001 Dec;81(6):3308-23
references.pmid: 11720994
references.citation: Diabetologia. 2009 May;52(5):739-51
references.pmid: 19288076
references.citation: Diabetes. 2006 Feb;55(2):441-51
references.pmid: 16443779
references.citation: Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):248-53
references.pmid: 9874804
references.citation: Biochem Biophys Res Commun. 2009 Jul 31;385(3):291-5
references.pmid: 19426714
references.citation: Diabetes. 1992 Apr;41(4):438-43
references.pmid: 1318855
references.citation: Am J Physiol Endocrinol Metab. 2003 Jul;285(1):E138-54
references.pmid: 12644446