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A microfluidic system for monitoring glucagon secretion from human pancreatic islets of Langerhans.

ABSTRACT: Glucagon is a 29-amino acid peptide released from α-cells within pancreatic islets of Langerhans to help raise blood glucose levels. While a plethora of methodologies have been developed for quantitative measurement of insulin released from islets, such methods are not well developed for glucagon despite its importance in blood sugar regulation. In this work, a simple yet robust microfluidic device was developed for holding human pancreatic islets and perfuse them with glucose. The perfusate was collected into 2 min fractions and glucagon quantified using a homogeneous time-resolved Förster resonance energy transfer (TR-FRET) sandwich immunoassay. Simulation of fluid flow within the microfluidic device indicated the device produced low amounts of shear stress on islets, and characterization of the flow with standard glucagon solutions revealed response times within 2 fractions (<4 min). Results with human islets from multiple donors demonstrated either a "burst" of glucagon or a "sustained" glucagon release across the entire period of stimulation. The simplicity, yet robustness, of the device and method is expected to appeal to a number of researchers examining pancreatic islet physiology.

SUBMITTER: Eaton WJ

PROVIDER: S-EPMC8375491 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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A microfluidic system for monitoring glucagon secretion from human pancreatic islets of Langerhans.

Eaton Wesley J WJ Roper Michael G MG

Analytical methods : advancing methods and applications 20210726 32

Glucagon is a 29-amino acid peptide released from α-cells within pancreatic islets of Langerhans to help raise blood glucose levels. While a plethora of methodologies have been developed for quantitative measurement of insulin released from islets, such methods are not well developed for glucagon despite its importance in blood sugar regulation. In this work, a simple yet robust microfluidic device was developed for holding human pancreatic islets and perfuse them with glucose. The perfusate was ...[more]

PMID: 34308945

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Project description:ObjectivePancreatic islets are nutrient sensors that regulate organismal blood glucose homeostasis. Glucagon release from the pancreatic α-cell is important under fasted, fed, and hypoglycemic conditions, yet metabolic regulation of α-cells remains poorly understood. Here, we identified a previously unexplored role for physiological levels of leucine, which is classically regarded as a β-cell fuel, in the intrinsic regulation of α-cell glucagon release.MethodsGcgCreERT:CAMPER and GcgCreERT:GCaMP6s mice were generated to perform dynamic, high-throughput functional measurements of α-cell cAMP and Ca2+ within the intact islet. Islet perifusion assays were used for simultaneous, time-resolved measurements of glucagon and insulin release from mouse and human islets. The effects of leucine were compared with glucose and the mitochondrial fuels 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH, non-metabolized leucine analog that activates glutamate dehydrogenase), α-ketoisocaproate (KIC, leucine metabolite), and methyl-succinate (complex II fuel). CYN154806 (Sstr2 antagonist), diazoxide (KATP activator, which prevents Ca2+-dependent exocytosis from α, β, and δ-cells), and dispersed α-cells were used to inhibit islet paracrine signaling and identify α-cell intrinsic effects.ResultsMimicking the effect of glucose, leucine strongly suppressed amino acid-stimulated glucagon secretion. Mechanistically, leucine dose-dependently reduced α-cell cAMP at physiological concentrations, with an IC50 of 57, 440, and 1162 μM at 2, 6, and 10 mM glucose, without affecting α-cell Ca2+. Leucine also reduced α-cell cAMP in islets treated with Sstr2 antagonist or diazoxide, as well as dispersed α-cells, indicating an α-cell intrinsic effect. The effect of leucine was matched by KIC and the glutamate dehydrogenase activator BCH, but not methyl-succinate, indicating a dependence on mitochondrial anaplerosis. Glucose, which stimulates anaplerosis via pyruvate carboxylase, had the same suppressive effect on α-cell cAMP but with lower potency. Similarly to mouse islets, leucine suppressed glucagon secretion from human islets under hypoglycemic conditions.ConclusionsThese findings highlight an important role for physiological levels of leucine in the metabolic regulation of α-cell cAMP and glucagon secretion. Leucine functions primarily through an α-cell intrinsic effect that is dependent on glutamate dehydrogenase, in addition to the well-established α-cell regulation by β/δ-cell paracrine signaling. Our results suggest that mitochondrial anaplerosis-cataplerosis facilitates the glucagonostatic effect of both leucine and glucose, which cooperatively suppress α-cell tone by reducing cAMP.

Dataset Information

A microfluidic system for monitoring glucagon secretion from human pancreatic islets of Langerhans.

Publications

A microfluidic system for monitoring glucagon secretion from human pancreatic islets of Langerhans.

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