Profiling Glucose-Stimulated and M3 Receptor-Activated Insulin Secretion Dynamics from Islets of Langerhans Using an Extended-Lifetime Fluorescence Dye

Abstract

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 Ca²⁺ oscillations within islets can be entrained by pulses of a cholinergic agonist, carbachol (CCh). Although these proxy measurements of Ca²⁺ 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.

Figure 1

Microfluidic chip, flow profile, and pulse train characterization. (A) Perfusion solutions or insulin standards were delivered to inlets 1 and 2 for islet experiments or calibrations, respectively, and passed through the closed islet chamber (green dot). The two remaining inlets were used to deliver Ab_Ins and Ins* as noted. The reagents mixed and equilibrated in the 160 mm long mixing channel before fluorescence measurement at the detection point (blue dot). Black arrows indicate the direction of flow. (B) Flow of SeTau-647 was initiated from at 0 min, while signal was measured at the detection point. The x-axis break at 0.5 min is for ease in viewing. (C) The blue trace (right axis) shows the programmed flow rates of SeTau-647 delivered from inlet 2. The black trace (left axis) is the fluorescence signal at the islet chamber during a pulsing experiment. A 53 ± 3% pulse attenuation was observed at the islet chamber.

Figure 2

Competitive immunoassay improvement with squaraine rotaxane fluorophore. (A) Calculated calibration curves using Ins*_Cy5 and Ins*_SeTau-647 were computed using their photophysical properties and mathematically calculated B/F ratios as explained in the text and Supporting Information. (B) Experimental calibration curves generated with 24 nM Ab_Ins and 24 nM of either Ins*_Cy5 or Ins*_SeTau-647. Δ⟨r⟩ values in panels A and B are taken as relative to the anisotropy at 0 nM. SeTau-647 is shown to produce improved assay range from both calculated (panel A) and experimental (panel B) results.

Figure 3

Online GSIS measurement from single and grouped murine islets. In both experiments, the black trace (left y-axis) is the insulin secretion rate per islet, whereas the blue profile (right axis) is the glucose stimulus. (A) A solution of 11 mM glucose was delivered for 40 min to a single murine islet during which biphasic, pulsatile insulin release was observed. (B) A group of five murine islets showed biphasic insulin secretion release during the 25 min exposure to 12 mM glucose.

Figure 4

Entraining effect of periodic CCh pulses on GSIS in murine islets. In both panels, five murine islets were perfused with a constant 11 mM glucose concentration. (A) A train of five CCh pulses were delivered for 30 s each, every 5 min, as denoted by an “×”. The numbered insulin peaks correlate to those mentioned in the text. (B) A representative control experiment is shown with flow switches denoted by “×”. Because no CCh was present, no large pulses of insulin were observed.