Abstract
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 (Δψ(p)) indicator in combination with indicators of cytoplasmic free Ca(2+) ([Ca(2+)](c)), mitochondrial membrane potential (Δψ(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 β-cells. Elevated glucose caused oscillations in plasma membrane potential and cytoplasmic free Ca(2+) 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 2.8 mM glucose. Increased glucose and pyruvate each produced a concentration-dependent mitochondrial hyperpolarization. The causal relationships between pairs of parameters (Δψ(p) and [Ca(2+)](c), Δψ(p) and NAD(P)H, matrix ATP and [Ca(2+)](c), and Δψ(m) and [Ca(2+)](c)) were investigated at single cell level. It is concluded that, in these β-cells, depolarizing oscillations in Δψ(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 Δψ(p) oscillatory mechanism is initiated.
Highlights
Plasma membrane potential (PMP) oscillations play a key role in glucose-stimulated insulin secretion
We have employed a novel fluorescent plasma membrane potential (⌬p) indicator in combination with indicators of cytoplasmic free Ca2؉ ([Ca2؉]c), mitochondrial membrane potential (⌬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 -cells
The anionic plasma membrane potential indicator (PMPI) [19] readily detects and resolves oscillations in these cells separated by 20 s or more, and we have exploited PMPI together with fluorescent probes of ⌬m, [Ca2ϩ]c, NAD(P)H, and matrix ATP and image analysis to conclude that these clonal -cells can maintain pyruvate-driven oscillations depolarizing ⌬p and elevating [Ca2ϩ]c that are not initiated by an increase in mitochondrial “function” (i.e. raised ⌬m, NAD(P)H, or matrix ATP)
Summary
Plasma membrane potential (PMP) oscillations play a key role in glucose-stimulated insulin secretion. We exploit a combination of fluorescent techniques to monitor changes in plasma membrane potential (⌬p), mitochondrial membrane potential ( ⌬m), cytoplasmic free Ca2ϩ ([Ca2ϩ]c), matrix ATP, and NAD(P)H reduction in the presence of glucose or pyruvate to investigate these questions in INS-1 832/13 cells. The anionic plasma membrane potential indicator (PMPI) [19] readily detects and resolves oscillations in these cells separated by 20 s or more, and we have exploited PMPI together with fluorescent probes of ⌬m, [Ca2ϩ]c, NAD(P)H, and matrix ATP and image analysis to conclude that these clonal -cells can maintain pyruvate-driven oscillations depolarizing ⌬p and elevating [Ca2ϩ]c that are not initiated by an increase in mitochondrial “function” (i.e. raised ⌬m, NAD(P)H, or matrix ATP). Small downstream changes can be detected, but the implication is that regardless of substrate, the mitochondria may be required to exceed a critical bioenergetic threshold to allow these cells to release insulin and that once this threshold is surpassed, an autonomous oscillatory mechanism is initiated
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have