Plasma Membrane Potential Oscillations in Insulin Secreting Ins-1 832/13 Cells Do Not Require Glycolysis and Are Not Initiated by Fluctuations in Mitochondrial Bioenergetics

Oscillations in plasma membrane potential play a central role in glucose-induced insulin secretion from pancreatic beta-cells and related insulinoma cell lines. We have employed a novel fluorescent plasma membrane potential (Delta psi(p)) indicator in combination with indicators of cytoplasmic free Ca2+ ([Ca2+](c)), mitochondrial membrane potential (Delta psi(m)), matrix ATP concentration, and NAD(P)H fluorescence to investigate the role of mitochondria in the generation of plasma membrane potential oscillations in clonal INS-1 832/13 beta-cells. Elevated glucose caused oscillations in plasma membrane potential and cytoplasmic free Ca2+ concentration over the same concentration range required for insulin release, although considerable cell-to-cell heterogeneity was observed. Exogenous pyruvate was as effective as glucose in inducing oscillations, both in the presence and absence of 2.8mM glucose. Increased glucose and pyruvate each produced a concentration-dependent mitochondrial hyperpolarization. The causal relationships between pairs of parameters (Delta psi(p) and [Ca2+](c), Delta psi(p) and NAD(P)H, matrix ATP and [Ca2+](c), and Delta psi(m) and [Ca2+](c)) were investigated at single cell level. It is concluded that, in these beta-cells, depolarizing oscillations in Delta psi(p) are not initiated by mitochondrial bioenergetic changes. Instead, regardless of substrate, it appears that the mitochondria may simply be required to exceed a critical bioenergetic threshold to allow release of insulin. Once this threshold is exceeded, an autonomous Delta psi(p) oscillatory mechanism is initiated.