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 K(ATP) 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, DELTApsi(m) (monitored using rhodamine 123 fluorescence), hyperpolarized with a latency of 49 +/- 6 s (n = 17), and the [Ca2+], 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 DELTApsi(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 K(ATP) 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.