Abstract
Studying the response of islet cells to glucose stimulation is important for understanding cell function in healthy and disease states. Most functional assays are performed on whole islets or cell populations, resulting in averaged observations and loss of information at the single cell level. We demonstrate methods to examine calcium fluxing in individual cells of intact islets in response to multiple glucose challenges. Wild-type mouse islets predominantly contained cells that responded to three (out of three) sequential high glucose challenges, whereas cells of diabetic islets (db/db or NOD) responded less frequently or not at all. Imaged islets were also immunostained for endocrine markers to associate the calcium flux profile of individual cells with gene expression. Wild-type mouse islet cells that robustly fluxed calcium expressed β cell markers (INS/NKX6.1), whereas islet cells that inversely fluxed at low glucose expressed α cell markers (GCG). Diabetic mouse islets showed a higher proportion of dysfunctional β cells that responded poorly to glucose challenges. Most of the failed calcium influx responses in β cells were observed in the second and third high glucose challenges, emphasizing the importance of multiple sequential glucose challenges for assessing the full function of islet cells. Human islet cells were also assessed and showed functional α and β cells. This approach to analyze islet responses to multiple glucose challenges in correlation with gene expression assays expands the understanding of β cell function and the diseased state.
Highlights
Techniques to assess pancreatic β cell function are required for developing β cell replacement therapies for diabetics
Towards the goal of assessing the function of healthy and diseased islets, a method was developed to image calcium flux of whole islets and individual islet cells in intact islets followed by fixation and immunostaining to examine marker expression profiles of calcium-imaged cells (Fig 1A)
Images were collected during glucose challenge to generate image stacks that were used to quantify the calcium signal at the population and single cell level over time
Summary
Techniques to assess pancreatic β cell function are required for developing β cell replacement therapies for diabetics. E.g. human β cells derived from the directed differentiation of pluripotent stem cells, need to be validated by various methods to assess physiological function. Histology, transcriptional and proteomic assays, and glucose stimulated insulin secretion (GSIS) assays are widely used to assess β cells or islets [1,2,3,4,5]. A major limitation of these techniques is the lack of temporal data, with regards to the PLOS ONE | DOI:10.1371/journal.pone.0122044. Islet Calcium Influx and Marker Correlation in Single Cell Level A major limitation of these techniques is the lack of temporal data, with regards to the PLOS ONE | DOI:10.1371/journal.pone.0122044 April 8, 2015
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