44-OR: Human Pseudo Islets Reveal Differential Effects of Gq Signaling in ß Cells in Low and High Glucose

Abstract

Efforts to advance our understanding of human islet biology are hindered by a paucity of approaches to effectively manipulate gene expression in human islets. To address this, we developed a pseudoislet system of human islet dispersion and reaggregation via modified hanging drop with 2500 cells/drop to generate 150-200 μm diameter pseudoislets (n=9 normal donors; 26-73 years). Pseudoislet morphology, dithizone uptake, and expression of markers of α and/or β cell identity (PDX1, NKX6.1, ARX, MAFB, NKX2.2, and PAX6) were similar to native islets; in paired comparisons from the same donor, pseudoislets had slightly fewer β cells and more α cells (53% β, 44% α, 3% δ cells) than native islets (62% β, 35% α, 3% δ cells; p<0.05 β and α cells). In perifusion, pseudoislets were similar to native islets in biphasic glucose-stimulated and cAMP-potentiated insulin secretion as well as in epinephrine and low glucose stimulation of and high glucose inhibition of glucagon release. Adenoviral-transduced dispersed human islet cells (>90% β cell transduction) formed functioning pseudoislets similar to untransduced controls. Using this approach, we virally expressed hM3Dq, a Gq-coupled designer receptor exclusively activated by a designer drug (DREADD), which selectively responds to the otherwise inert ligand clozapine-n-oxide (CNO) and signals through PLC/IP3. In low glucose (2mM G + 10μM CNO), hM3Dq pseudoislets secreted more insulin than control mCherry pseudoislets (19.2 vs. 1.0 ng/100IEQ/hr; p<0.0001). In contrast, hM3Dq and control pseudoislets had similar insulin secretion in high glucose (11mM G + 10μM CNO; 24.8 vs. 23.2 ng/100IEQ/hr; p>0.05), indicating that activation of Gq signaling does not potentiate insulin secretion in high glucose. These data demonstrate how manipulation of gene expression in pseudoislets can provide insight into human islet biology and suggest that activation of Gq signaling in β cells elicits robust insulin secretion that appears to be uncoupled from glucose metabolism. Disclosure J.T. Walker: None. R. Haliyur: None. R. Aramandla: None. G. Poffenberger: None. H.A. Nelson: None. J. Luchsinger: None. P. Wang: None. A. Garcia-Ocaña: None. M. Brissova: None. A.C. Powers: None.