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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

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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 1

Pseudoislets resemble native human islets in morphology, cell composition, and function.

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Pseudoislets resemble native human islets in morphology, cell compositio...
(A) Schematic of pseudoislet formation. (B) Bright-field images showing the morphology of native islets and pseudoislets. Scale bar: 200 μm. (C) Relative frequency plot of islet diameter comparing hand-picked native islets with pseudoislets from the same donor. (D) Dithizone (DTZ) uptake of native islets and pseudoislets. Scale bar: 200 μm. (E) Insulin and glucagon content normalized to islet volume expressed in islet equivalents (IEQs); 1 IEQ corresponds to an islet with a diameter of 150 μm; n = 5 donors; P > 0.05. (F) Confocal images of native islets and pseudoislets stained for insulin (INS; β cells), glucagon (GCG; α cells), and somatostatin (SOM; δ cells). Scale bar: 50 μm. (G) Quantification of relative endocrine cell composition of native islets and pseudoislets; n = 4 donors; P > 0.05. Insulin (H) and glucagon (I) secretory response to various secretagogues measured by perifusion of native islets and pseudoislets from the same donor (n = 5). G 5.6, 5.6 mM glucose; G 16.7, 16.7 mM glucose; G 16.7 + IBMX 100, 16.7 mM glucose with 100 μM isobutylmethylxanthine (IBMX); G1.7 + Epi 1, 1.7 mM glucose and 1 μM epinephrine; KCl 20, 20 mM potassium chloride (KCl). Wilcoxon matched-pairs signed-rank test was used to analyze statistical significance in E and G. H and I were analyzed by 2-way ANOVA; P > 0.05. The area under the curve for each secretagogue was compared by 1-way ANOVA with Dunn’s multiple comparison test (Supplemental Figure 1, E–H and J–M). Data are represented as mean ± SEM.

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