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 β-cells and related insulinoma cell lines. We have employed a novel fluorescent plasma membrane potential (ψp) indicator in combination with indicators of cytoplasmic free Ca2+ ([Ca2+]c), mitochondrial membrane potential (ψm), 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 β-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 - ψp and [Ca2+]c, ψp and NAD(P)H, matrix ATP and [Ca2+]c, and ψm and [Ca2+]c were investigated at single cell level. It is concluded that, in these β-cells, depolarizing oscillations in ψp 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 ψp oscillatory mechanism is initiated.