Abstract
The temporal relationship of glucose-induced increases in cytoplasmic pH (pHi) and cytoplasmic free Ca2+ was studied in single mouse pancreatic beta-cells and suspensions of clonal beta-cells (HIT). In both preparations of cells the increase in pHi preceded the cytoplasmic free Ca2+ increase. Therefore the alkalinization cannot be a consequence of the Ca2+ influx. A potential metabolic mechanism for the increase in pHi, involving stimulation of pyruvate transport and oxidation, was demonstrated in a model system of liver mitochondria incubated with pyruvate, ATP, and hexokinase to which glucose was then added to initiate ATP use. The involvement of this mechanism in beta-cells is suggested by the observation that the alkalinization was prevented in most cells by incubation with 3-hydroxycyanocinnamate, a mitochondrial pyruvate transport inhibitor. On the other hand, the inhibited cells exhibited normal Ca2+ responses to glucose stimulation. This indicates that neither pyruvate metabolism nor the alkalinization is of critical importance for the Ca2+ signal, though pyruvate oxidation or its metabolites may be important in downstream regulation of secretion.
Highlights
From the $Rolf Luft Center for DiabetesResearch, Department of Endocrinology, Karolinska Institute, Box 60 500, Karolinska Hospital, S-10401 Stockholm, Sweden a n d the $Diabetes a n d Metabolism Unit, Evans Department of Medicine, and Wepartmentof Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
When single mouse pancreatic p-cells were stimulated with glucose, there was an initial transienitncrease in pH, (Fig. 1)
In cells loaded with both the Ca2+indicator fura-2andthe pHindicator BCECF, it could be clearly seen that the [Ca2+1r,ise occurred after the alkalinization, as illustrated Finig. 2
Summary
An increase in mechanism for the increase in pH, involving stimda- [Ca2+l,may cause an increase in pHi, as demonstrated in thytion of pyruvate transport and oxidation, was demonstrated in a model system of liver mitochondria incubatedwithpyruvate, ATP, andhexokinasetowhich glucose was added to initiaAteTP use. The involvement of this mechanism in &cells is suggested by the observation that the alkalinization was prevented in mocytes and fibroblasts [23]. Most cells by incubationwith3-hydroxycyanocinnamate, a mitochondrial pyruvate transport inhibitor
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