Temporal sequence of metabolic and ionic events in glucose-stimulated clonal pancreatic beta-cells (HIT).
Stimulation of insulin release by glucose requires increased metabolism of glucose and a rise in cytosolic free Ca2+ in the pancreatic beta-cell. It is accompanied by increases in respiratory rate, pyridine and flavin nucleotide reduction state, intracellular pH and the ATP/ADP ratio. To test alternative proposals of the regulatory relationships among free Ca2+, mitochondrial metabolism and cellular energy state, we determined the temporal sequence of these metabolic and ionic changes following addition of glucose to clonal pancreatic beta-cells (HIT). Combined measurements of the native fluorescence of reduced pyridine nucleotides and oxidized flavin, intracellular pH, and free Ca2+ were performed together with simultaneous measurement of O2 tension or removal of samples for assay of the ATP/ADP ratio. The initial changes were detected in three phases. First, decreases occurred in the ATP/ADP ratio (<3 s) and increases in pyridine (2 +/- 1 s) and flavin (2 +/- 1 s) nucleotide reduction. Next, increases in the O2 consumption rate (20 +/- 5 s), the ATP/ADP ratio (29 +/- 12 s) and internal pH (48 +/- 5 s) were observed. Finally, cytosolic free Ca2+ rose (114 +/- 10 s). Maximal changes in the ATP/ADP ratio, O2 consumption and pyridine and flavin nucleotide fluorescence preceded the beginning of the Ca2+ change. These relationships are consistent with a model in which phosphorylation of glucose is the initial event which generates the signals that lead to an increase in respiration, a rise in the ATP/ADP ratio and finally influx of Ca2+. Our results indicate that Ca2+ does not function as the initiator of increased mitochondrial respiration.
Civelek
V N
VN
Diabetes and Metabolism Unit, Evans Department of Medicine, Boston University Medical Center, MA 02118, USA.
Deeney
J T
JT
Kubik
K
K
Schultz
V
V
Tornheim
K
K
Corkey
B E
BE
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002999:Clone Cells; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010101:Oxygen Consumption; D010766:Phosphorylation; D015398:Signal Transduction
D007328:Insulin; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005947:Glucose; D002118:Calcium