Substrate-dependent changes in mitochondrial function, intracellular free calcium concentration and membrane channels in pancreatic beta-cells.
Microfluorimetric and patch-clamp techniques have been combined to determine the relationship between changes in mitochondrial metabolism, the activity of KATP channels and changes in intracellular free calcium concentration ([Ca2+]i) in isolated pancreatic beta-cells in response to glucose, ketoisocaproic acid (KIC) and the electron donor couple tetramethyl p-phenylenediamine (TMPD) and ascorbate. Exposure of cells to 20 mM glucose raised NAD(P)H autofluorescence after a delay of 28 +/- 1 s (mean +/- S.E.M., n = 30). The mitochondrial inner membrane potential, delta psi m (monitored using rhodamine 123 fluorescence), hyperpolarized with a latency of 49 +/- 6 s (n = 17), and the [Ca2+]i rose after 129 +/- 13 s (n = 5). The amplitudes of the metabolic changes were graded appropriately with glucose concentration over the range 2.5-20 mM. All variables responded to KIC with shorter latencies: NAD(P)H autofluorescence rose after a delay of 20 +/- 3 s (n = 5) and rhodamine 123 changed after 21 +/- 3 s (n = 6). The electron donor couple, TMPD with ascorbate, rapidly hyperpolarized delta psi m and raised [Ca2+]i. When [Ca2+]i was raised by sustained exposure to 20 mM glucose, TMPD had no further effect. TMPD also decreased whole-cell KATP currents and depolarized the cell membrane, measured with the perforated patch configuration. These data are consistent with a central role for mitochondrial oxidative phosphorylation in coupling changes in glucose concentration with the secretion of insulin.
Duchen
M R
MR
Department of Physiology, University College London, U.K.
Smith
P A
PA
Ashcroft
F M
FM
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D066298:In Vitro Techniques; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D008297:Male; D008564:Membrane Potentials; D051379:Mice; D008807:Mice, Inbred BALB C; D008928:Mitochondria / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013772:Tetramethylphenylenediamine / Q000494:pharmacology
D015221:Potassium Channels; D009243:NAD; D005947:Glucose; D013772:Tetramethylphenylenediamine; D001205:Ascorbic Acid; D002118:Calcium