Integrated human pseudoislet system and microfluidic platform demonstrate differences in GPCR signaling in islet cells
John T. Walker, … , Alvin C. Powers, Marcela Brissova
John T. Walker, … , Alvin C. Powers, Marcela Brissova
Published April 30, 2020
Citation Information: JCI Insight. 2020;5(10):e137017. https://doi.org/10.1172/jci.insight.137017.
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Resource and Technical Advance Endocrinology Metabolism

Integrated human pseudoislet system and microfluidic platform demonstrate differences in GPCR signaling in islet cells

Abstract

Pancreatic islets secrete insulin from β cells and glucagon from α cells, and dysregulated secretion of these hormones is a central component of diabetes. Thus, an improved understanding of the pathways governing coordinated β and α cell hormone secretion will provide insight into islet dysfunction in diabetes. However, the 3D multicellular islet architecture, essential for coordinated islet function, presents experimental challenges for mechanistic studies of intracellular signaling pathways in primary islet cells. Here, we developed an integrated approach to study the function of primary human islet cells using genetically modified pseudoislets that resemble native islets across multiple parameters. Further, we developed a microperifusion system that allowed synchronous acquisition of GCaMP6f biosensor signal and hormone secretory profiles. We demonstrate the utility of this experimental approach by studying the effects of Gi and Gq GPCR pathways on insulin and glucagon secretion by expressing the designer receptors exclusively activated by designer drugs (DREADDs) hM4Di or hM3Dq. Activation of Gi signaling reduced insulin and glucagon secretion, while activation of Gq signaling stimulated glucagon secretion but had both stimulatory and inhibitory effects on insulin secretion, which occur through changes in intracellular Ca2+. The experimental approach of combining pseudoislets with a microfluidic system allowed the coregistration of intracellular signaling dynamics and hormone secretion and demonstrated differences in GPCR signaling pathways between human β and α cells.

Authors

John T. Walker, Rachana Haliyur, Heather A. Nelson, Matthew Ishahak, Gregory Poffenberger, Radhika Aramandla, Conrad Reihsmann, Joseph R. Luchsinger, Diane C. Saunders, Peng Wang, Adolfo Garcia-Ocaña, Rita Bottino, Ashutosh Agarwal, Alvin C. Powers, Marcela Brissova

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Figure 3

Gi activation reduces insulin and glucagon secretion.

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Gi activation reduces insulin and glucagon secretion. (A) Schematic of i...
(A) Schematic of incorporation of efficient viral transduction into pseudoislet approach. (B) Schematic of the Gi-coupled GPCR signaling pathway. CNO, clozapine-N-oxide; AC, adenylyl cyclase; ATP, adenosine triphosphate; GIRK, G protein–coupled inwardly rectifying potassium channel; K+, potassium ion. (C) Dynamic insulin secretion assessed by macroperifusion in response to low glucose (G 2, 2 mM glucose; white), glucose ramp (G 7, 7 mM; G 11, 11 mM; G 20, 20 mM glucose; gray), and KCl-mediated depolarization (KCl 20, 20 mM potassium chloride in the presence of G 2 or G 11; yellow) in the absence (blue trace) or presence of CNO (red trace); n = 4 donors/each. 10 μM CNO was added after the first period of 2 mM glucose, as indicated by a vertical red arrow and then continuously administered for the duration of the experiment (red trace). Note the split of y axis to visualize differences between traces at G 2 ± CNO. (D–F) Insulin secretion was integrated by calculating the area under the curve (AUC) for response to the low glucose (white), glucose ramp (gray), and KCl-mediated depolarization (yellow). Baseline was set to the average value of each trace from 0 to 21 minutes (before CNO addition). (G–J) Glucagon secretion was analyzed in parallel with insulin as described above. Insulin and glucagon secretory traces in C and G, respectively, were compared in the absence versus presence of CNO by 2-way ANOVA; ****P < 0.0001 for both insulin and glucagon secretion. Area under the curve of insulin (D–F) and glucagon responses (H–J) to low glucose, glucose ramp, and KCl-mediated depolarization were compared in the absence versus presence of CNO by Mann-Whitney test; *P < 0.05. Data are represented as mean ± SEM.