A testable """"""""metabolic hypothesis"""""""" for the postprandial induction of insulin secretion is that B cell metabolism of nutrient fuels, especially glucose, -(1)-> alteration of intracellular intermediates -(2)-> closure of metabolically regulated K+ channels _(3)-> cell depolarization -(4)-> action potential initiation and the opening of voltage dependent Ca2+ channels - (5)-> Ca2+ entry -(6)-> insulin granule exocytosis. Over the past five years important advances have been made in identifying """"""""molecular players"""""""" in this hypothesis including an ATP1 sensitive K+ channel, whose closure depolarizes the cell; voltage dependent Ca2+, and K+ channels, which probably contribute to the action potential; and increases in cytosolic Ca2+ under conditions where B cells depolarize. We propose to further verify this hypothesis by investigating several dynamic links (4- 6) which connect cell depolarization to granule exocytosis by simultaneously applying several new """"""""integrative"""""""" techniques to single normal adult human islet B cells harvested from cadaver donors. These techniques are """"""""perforated patch"""""""" current or voltage clamp recording; microspectrofluometry, using Ca2+- sensitive dyes; and membrane capacitance measurements by """"""""phase detection"""""""" techniques. First we shall examine the ionic channel currents underlying patterns of metabolite induced electrical activity, especially Ca2+ channels. Second, we shall correlate changes in cytosolic Ca2+ with patterns of electrical activity induced by nutrient fuel secretogogues. Third, we shall attempt to correlate patterns of electrical activity and Ca2+ entry with patterns of insulin granule exocytosis as reflected by changes in membrane capacitance. The last approach will require us to establish optimal conditions for insulin secretion using the """"""""reverse hemolytic plaque assay"""""""" and to verify that capacitance changes seen reflect real-time incorporation of insulin granule membrane into the cell surface. The experiments should further our understanding of stimulus-secretion coupling in the normal human B cell and serve as a basis for studies on the pathophysiology of diminished insulin secretion in non-insulin dependent diabetes mellitus.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK037380-08
Application #
2140084
Study Section
Physiology Study Section (PHY)
Project Start
1986-07-01
Project End
1996-06-30
Budget Start
1994-07-01
Budget End
1996-06-30
Support Year
8
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Barnes-Jewish Hospital
Department
Type
DUNS #
City
Saint Louis
State
MO
Country
United States
Zip Code
63110
Misler, Stanley; Gillis, Kevin D (2012) Modes of exocytosis and electrogenesis underlying canine biphasic insulin secretion. Front Biosci (Elite Ed) 4:669-76
Silva, Amelia M; Dickey, Adam S; Barnett, David W et al. (2009) Ion channels underlying stimulus-exocytosis coupling and its cell-to-cell heterogeneity in beta-cells of transplantable porcine islets of Langerhans. Channels (Austin) 3:91-100
Misler, Stanley; Zhou, Zhuan; Dickey, Adam S et al. (2009) Electrical activity and exocytotic correlates of biphasic insulin secretion from beta-cells of canine islets of Langerhans: contribution of tuning two modes of Ca2+ entry-dependent exocytosis to two modes of glucose-induced electrical activity. Channels (Austin) 3:181-93
Misler, Stanley (2009) Unifying concepts in stimulus-secretion coupling in endocrine cells and some implications for therapeutics. Adv Physiol Educ 33:175-86
Misler, Stanley; Silva, Amelia M; Barnett, David et al. (2009) Phasic and tonic modes of depolarization-exocytosis coupling in beta-cells of porcine islets of Langerhans. Channels (Austin) 3:101-9
Luciani, Dan S; Misler, Stanley; Polonsky, Kenneth S (2006) Ca2+ controls slow NAD(P)H oscillations in glucose-stimulated mouse pancreatic islets. J Physiol 572:379-92
Silva, Amelia M; Liu-Gentry, June; Dickey, Adam S et al. (2005) alpha-Latrotoxin increases spontaneous and depolarization-evoked exocytosis from pancreatic islet beta-cells. J Physiol 565:783-99
Misler, Stanley; Dickey, Adam; Barnett, David W (2005) Maintenance of stimulus-secretion coupling and single beta-cell function in cryopreserved-thawed human islets of Langerhans. Pflugers Arch 450:395-404
Johnson, James D; Ahmed, Noreen T; Luciani, Dan S et al. (2003) Increased islet apoptosis in Pdx1+/- mice. J Clin Invest 111:1147-60
Johnson, James D; Misler, Stanley (2002) Nicotinic acid-adenine dinucleotide phosphate-sensitive calcium stores initiate insulin signaling in human beta cells. Proc Natl Acad Sci U S A 99:14566-71

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