Affiliation:
1. Diabetes and Metabolism Unit, Evans Department of Medicine, U.S.A.
2. Department of Biochemistry, Boston University Medical Center, Boston, MA 02118, U.S.A.
Abstract
Stimulation of insulin release by glucose requires increased metabolism of glucose and a rise in cytosolic free Ca2+ in the pancreatic β-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 β-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.
Subject
Cell Biology,Molecular Biology,Biochemistry
Cited by
61 articles.
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