Fluorescence recovery after photobleaching reveals regulation and distribution of connexin36 gap junction coupling within mouse islets of Langerhans

Abstract

Key pointsr Gap junctions provide electrical coupling that is critical to the function of pancreatic islets.Disruptions to connexin36 (Cx36) have been suggested to occur in diabetes.r No accurate and non-invasive technique has yet been established to quantify changes in Cx36 gap junction coupling in the intact islet.r This study developed fluorescence recovery after photobleaching (FRAP) as a non-invasive technique for quantifying Cx36 gap junction coupling in living islets.r The study identified treatments that modulate gap junction coupling, confirmed that the cellular distribution of coupling throughout the islet is highly heterogeneous and confirmed that α cells and β cells do not form functional Cx36 gap junctions.r This technique will enable future studies to examine the regulation of Cx36 gap junction coupling and its disruption in diabetes, and to uncover potential novel therapeutic targets associated with gap junction coupling. AbstractThe pancreatic islets are central to the maintenance of glucose homeostasis through insulin secretion. Glucose-stimulated insulin secretion is tightly linked to electrical activity in β cells within the islet. Gap junctions, composed of connexin36 (Cx36), form intercellular channels between β cells, synchronizing electrical activity and insulin secretion. Loss of gap junction coupling leads to altered insulin secretion dynamics and disrupted glucose homeostasis. Gap junction coupling is known to be disrupted in mouse models of pre-diabetes. Although approaches to measure gap junction coupling have been devised, they either lack cell specificity, suitable quantification of coupling or spatial resolution, or are invasive. The purpose of this study was to develop fluorescence recovery after photobleaching (FRAP) as a technique to accurately and robustly measure gap junction coupling in the islet. The cationic dye Rhodamine 123 was used with FRAP to quantify dye diffusion between islet β cells as a measure of Cx36 gap junction coupling. Measurements in islets with reduced Cx36 verified the accuracy of this technique in distinguishing between distinct levels of gap junction coupling. Analysis of individual cells revealed that the distribution of coupling across the islet is highly heterogeneous. Analysis of several modulators of gap junction coupling revealed glucose-and cAMP-dependent modulation of gap junction coupling in islets. Finally, FRAP was used to determine cell population specific coupling, where no functional gap junction coupling was observed between α cells and β cells in the islet. The results of this study show FRAP to be a robust technique which provides the cellular resolution to quantify the distribution and regulation of Cx36 gap junction coupling in specific cell populations within the islet. Future studies utilizing this technique may elucidate the role of gap junction coupling in the progression of diabetes and identify mechanisms of gap junction regulation for potential therapies.

Beta cell connectivity in pancreatic islets: a type 2 diabetes target?

“…It is unclear how GJs could account for the long-range functional connections that project between distant cells, as practically all beta cells express Cx36 protein, meaning that communication should encompass even close neighbors [65,72]. However, heterogeneity exists in fluorescence recovery after photobleaching (FRAP) within islets [73], suggesting that connectivity patterns between individual beta cells may at least reflect differences in functional GJ coupling. As proposed above, this may lead to the formation of linear groups of cells, tightly interconnected in three dimensions between one another, but (relatively) isolated from neighboring cells outside the train, thus forming a conduit for the passage of ionic (Ca 2? )…”

“…By contrast, GLP-1-regulated connectivity was normal, suggesting that ADCY5 is unlikely to link incretin signaling to cAMP generation and beta cell communication. Thus, ADCY5 preferentially affects glucose-induced human islet dynamics, possibly through cAMP, which has been shown to increase GJ conductance and trafficking [22,73,150], although this has only been so far demonstrated in rodent tissues.…”

Beta-cell hubs maintain Ca2+ oscillations in human and mouse islet simulations

“…This could, for example, involve FRAP-based monitoring of functionally identified hubs 64 or laser capture microdissection of hubs 67 followed by single cell RNA-seq. 68 …”

Low Level Pro-inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide-mediated Protein Kinase Cδ

“…Fluorescence Recovery after Photobleaching (FRAP)-FRAP was used to quantify the extent of Cx36 gap junction coupling, as previously described (43). Briefly, islets were cultured in MatTek dishes (MatTek Corp., Ashland, MA) coated with CellTak (BD Biosciences, San Jose, CA) and stained with 12.5 M rhodamine 123 (Sigma) for 30 min at 37°C.…”