Single islet beta-cell stimulation by nutrients: relationship between pyridine nucleotides, cytosolic Ca2+ and secretion.

Abstract

It is generally believed that the initiation of insulin secretion by nutrient stimuli necessitates the generation of metabolic coupling factors, leading to membrane depolarization and the gating of voltage‐sensitive Ca2+ channels. To establish this sequence of events, the kinetics of endogenous fluorescence of reduced pyridine nucleotides [NAD(P)H], reflecting nutrient metabolism, were compared to those of cytosolic calcium ([Ca2+]i) rises in single cultured rat islet beta‐cells. In preliminary experiments, the loss of quinacrine fluorescence from prelabelled cells was used as an indicator of secretion. This dye is concentrated in the acidic insulin‐containing secretory granules. Both glucose and 2‐ketoisocaproate (KIC) raised [Ca2+]i in a dose‐dependent manner. There was marked cellular heterogeneity in the [Ca2+]i response patterns. The two nutrient stimuli also increased NAD(P)H fluorescence, again showing cell‐to‐cell variations. In combined experiments, where the two parameters were measured in the same cell, the elevation of the NAD(P)H fluorescence preceded the rise in [Ca2+]i, confirming the statistical evaluation performed on separate cells. The application of two consecutive glucose challenges revealed coordinated changes in [Ca2+]i and NAD(P)H fluorescence. Finally, quinacrine secretion was stimulated by two nutrients with onset times similar to those recorded for [Ca2+]i elevations. These results clearly demonstrate that increased metabolism occurs during the lag period preceding Ca2+ influx via voltage‐sensitive Ca2+ channels, a prerequisite for the triggering of insulin secretion by nutrient stimuli.

Differential expression of E-cadherin at the surface of rat β-cells as a marker of functional heterogeneity

“…The amounts of both NCAM and integrins have thus been shown to differ from one b-cell to the next (Bosco et al 2000, Bernard-Kargar et al 2001, and functional heterogeneity has been documented by several groups (Salomon & Meda 1986, Stefan et al 1987, Pralong et al 1990, Soria et al 1991, van Schravendijk et al 1992. In the present study, the strongest labeling of E-cadherin was observed in the more central part of islets, corresponding to b-cells that have the higher insulin secretion activity (Stefan et al 1987).…”

Involvement of Per–Arnt–Sim (PAS) kinase in the stimulation of preproinsulin and pancreatic duodenum homeobox 1 gene expression by glucose

“…Exposure of ␤ cells to glucose also leads to increased concentrations of a number of molecules, which may conceivably interact with PAS domains (38) and affect a change in PASK tertiary structure and hence phosphorylation state. These include reduced pyridine nucleotides (39,40), ATP and ADP (11,41), and Ca 2ϩ (42), in addition to a range of glycolytic intermediates, amino acids (43), and fatty acids (44), as well as a decrease in O 2 tension (45) and membrane potential (46). Identification of those parameters that play a role in regulating PASK activity requires further investigation.…”

Alterations of insulin response to different β cell secretagogues and pancreatic vascular resistance induced by Nω‐nitro‐L‐arginine methyl ester

“…From the effect of D-NAME on both phases of fi cell response to the amino-acid, it can be concluded that a direct depolarizing effect could, again, account for a small part of the potentiation by L-NAME of insulin secretion induced by leucine. This amino acid is not a substrate for NO synthase but a-ketoisocaproate is able within seconds to increase intracellular levels of NADPH and Ca2+ (Pralong et al, 1990); both enzymatic co-factors, allowing an increased NO production, might thereby exert an inhibitory feed-back unmasked by L-NAME administration. Such an hypothesis is supported by the relatively low effectiveness of L-NAME in potentiating tolbutamide and KCl-induced insulin secretions.…”