Abstract
The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. β- and α-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing δ-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca2+ and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca2+ signaling among α-cells. The measurements allowed comparison of the phase relationship of Ca2+ oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets.
Highlights
The pancreatic islets of Langerhans are key regulators of glucose homeostasis
Isolated mouse islets were transduced with adenovirus expressing green fluorescent protein (GFP) or the red fluorescent protein mCherry conditionally controlled by the Rip2 promoter and the Tet-On 3G system and subsequently imaged with confocal microscopy
When islets with induced Rip2-mCherry expression were instead immunostained for glucagon only 6 % of the fluorescent cells showed both labels (Fig. 2b). mCherry expression is apparently a good predictor of β-cell identity since 90 % of the red cells stained for insulin and only 7 % for glucagon
Summary
The pancreatic islets of Langerhans are key regulators of glucose homeostasis. They contain four major types of endocrine cells: α-, β-, δ-, and PP-cells, secreting glucagon, insulin, somatostatin, and pancreatic polypeptide (PP), respectively [36, 45]. Insulin and glucagon pulsatility is typically disturbed in early stages of diabetes [27, 34, 40] but the molecular mechanisms that underlie the dysregulated secretion are unclear. PP was recently found to suppress glucagon secretion by direct effects on α-cells [2] and somatostatin inhibits both insulin and glucagon secretion [18]. Somatostatin pulsatility is in phase with that of insulin [20, Pflugers Arch - Eur J Physiol (2016) 468:1765–1777
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