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

EMBO J. 1990 Jan;9(1):53-60.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium / metabolism*
  • Cytosol / metabolism
  • Dose-Response Relationship, Drug
  • Glucose / pharmacology*
  • Insulin / metabolism*
  • Insulin Secretion
  • Islets of Langerhans / drug effects
  • Islets of Langerhans / physiology*
  • Keto Acids / pharmacology*
  • Male
  • NAD / metabolism*
  • NADP / metabolism*
  • Rats
  • Rats, Inbred Strains
  • Spectrometry, Fluorescence

Substances

  • Insulin
  • Keto Acids
  • NAD
  • NADP
  • alpha-ketoisocaproic acid
  • Glucose
  • Calcium