Pulsatile insulin
from pancreatic islets is crucial for glucose
homeostasis, but the mechanism behind coordinated pulsatility is still
under investigation. One hypothesis suggests that cholinergic stimulation
of islets by pancreatic ganglia resets these endocrine units, producing
synchronization. Previously, it was shown that intracellular Ca
2+
oscillations within islets can be entrained by pulses of
a cholinergic agonist, carbachol (CCh). Although these proxy measurements
of Ca
2+
provided insight into the synchronization mechanism,
measurement of insulin output would be more direct evidence. To this
end, a fluorescence anisotropy competitive immunoassay for online
insulin detection from single and grouped islets in a microfluidic
system was developed using a piezoelectric pressure-driven fluid delivery
system and a squaraine rotaxane fluorophore, SeTau-647, as the fluorescent
label for insulin. Due to SeTau-647 having a longer lifetime and higher
brightness compared to the previously used Cy5 fluorophore, a 45%
increase in the anisotropy range was observed with enhanced signal-to-noise
ratio (S/N) of the measurements. This new system was tested by measuring
glucose-stimulated insulin secretion from single and groups of murine
and human islets. Distinct islet entrainment of groups of murine islets
by pulses of CCh was also observed, providing further evidence for
the hypothesis that pulsatile output from the ganglia can synchronize
islet behavior. We expect that this relatively straightforward, homogeneous
assay can be widely used for examining not only insulin secretion
but other secreted factors from different tissues.
An LC-MS method using multiple reaction monitoring for quantitation of insulin, C-peptide, glucagon, and somatostatin secretion from human islet populations is described. A 5 min separation was developed using a 2.1 × 30 mm (i.d. x length) C18 column. The method was used to measure secretions from static incubations of human islets from 2 donors. Insulin and C-peptide matched well with reported values of these hormones.
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