In vivomonitoring of intracellular Ca²⁺dynamics in the pancreatic β-cells of zebrafish embryos

Abstract

Assessing the response of pancreatic islet cells to glucose stimulation is important for understanding β-cell function. Zebrafish are a promising model for studies of metabolism in general, including stimulus-secretion coupling in the pancreas. We used transgenic zebrafish embryos expressing a genetically-encoded Ca2+ sensor in pancreatic β-cells to monitor a key step in glucose induced insulin secretion; the elevations of intracellular [Ca2+]i. In vivo and ex vivo analyses of [Ca2+]i demonstrate that β-cell responsiveness to glucose is well established in late embryogenesis and that embryonic β-cells also respond to free fatty acid and amino acid challenges. In vivo imaging of whole embryos further shows that indirect glucose administration, for example by yolk injection, results in a slow and asynchronous induction of β-cell [Ca2+]i responses, while intravenous glucose injections cause immediate and islet-wide synchronized [Ca2+]i fluctuations. Finally, we demonstrate that embryos with disrupted mutation of the CaV1.2 channel gene cacna1c are hyperglycemic and that this phenotype is associated with glucose-independent [Ca2+]i fluctuation in β-cells. The data reveal a novel central role of cacna1c in β-cell specific stimulus-secretion coupling in zebrafish and demonstrate that the novel approach we propose – to monitor the [Ca2+]i dynamics in embryonic β-cells in vivo – will help to expand the understanding of β-cell physiological functions in healthy and diseased states.

Beta‐cell excitability and excitability‐driven diabetes in adult Zebrafish islets

“…; Lorincz et al. ); ex vivo recordings of these younger zebrafish islets clearly show cells activating independently, at thresholds between 5 and 20 mmol/L glucose. Absence of cell‐cell electrical coupling in zebrafish β ‐cells may explain, in part, the relatively lower glucose tolerance of zebrafish compared to mammals (Eames et al.…”

“…), and intracellular [Ca 2+ ] is glucose‐dependent in larval zebrafish islets (Lorincz et al. ). In the present study we have now characterized glucose‐dependence of intracellular calcium in adult zebrafish islets, and show that the glucose dependence of calcium oscillations is similar, although not identical, to mammalian islets.…”

“…7 | Iss. 11 | e14101 Page 5 (Lorincz et al 2018), the amino acids glutamine, alanine, and leucine caused no activation in the presence of threshold (8 mmol/L) glucose (Fig. 3D).…”

“…We recently showed that intracellular [Ca 2+ ] is glucose‐sensitive in embryonic zebrafish islets (Lorincz et al. ). Here we characterize the glucose‐sensitivity of intracellular [Ca 2+ ] in adult zebrafish islets, and show that it is similar to mammals, although unlike in mammalian islets (Bavamian et al.…”

“…However, direct assessment of zebrafish islet function is lacking, and whether alterations in excitability can drive persistent changes in glucose control in zebrafish, remains unknown. We recently showed that intracellular [Ca 2+ ] is glucose-sensitive in embryonic zebrafish islets (Lorincz et al 2018). Here we characterize the glucose-sensitivity of intracellular [Ca 2+ ] in adult zebrafish islets, and show that it is similar to mammals, although unlike in mammalian islets (Bavamian et al 2007), b-cells appear to function as independent units, such that Ca 2+ transients are not well-coupled between b-cells.…”