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 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.

Automated cellular stimulation with integrated pneumatic valves and fluidic capacitors

“…Fluorescence anisotropy was measured as has been described elsewhere. 21,22 Briefly, the microfluidic device was placed on the stage of an Eclipse TS-100 microscope (Nikon Instruments Inc., Melville, NY). Fluorescence excitation was from a 25 mW 635 nm laser (Coherent Inc. Santa Clara, CA) attenuated to 2 mW with a neutral density filter (Thorlabs Inc., Newton, NJ) and coupled into a multimode fiber-optic bundle (Ceramoptec, Sunnyvale, CA) by an achromatic fiber port collimator (PAF2S-7A, Thorlabs).…”

Advances in microfluidic chips based on islet hormone-sensing techniques

“…As anisotropy is highly determined by fluorophore lifetime, different fluorophores will generate different anisotropy ranges and varied detection resolution. Adablah, in the same group as Schrell, replaced the Cy5 with SeTAu-647 characterized by a longer lifetime and higher brightness, and the alteration resulted in a 45% increase in anisotropy range and a significant improvement in signal-to-noise ratio[ 54 ]. With the development of this technology, a highly integrated chip for separately sensing up to 12 islet secretomes was designed by Wang et al [ 55 ], this integration ensures the possibility of high-throughput single islet secretome monitoring, and the procedure for islet heterogeneity study is markedly simplified.…”

Microfluidic systems in clinical diagnosis

“…Glucose is metabolized in the liver, where it is converted to glycogen, there is a complex dynamic between the pancreas and the liver to achieve glucose homeostasis. The Roper group has investigated the secretion of insulin [95][96][97][98] glucagon [97,99] and small molecules [100] from islets of Langerhans, which are islands of endocrine cells in the pancreas; and glucose consumption [101] by HepG2 hepatocytes, which are epithelial liver cells. The authors stated that all of the systems used for characterization of the cell secretion characterization can be implemented in other laboratories, as the systems rely on robust optical detection.…”