Kenty Jennifer H R, Melton Douglas A
Department of Stem Cell and Regenerative Biology, Harvard University, Harvard Stem Cell Institute, Cambridge, Massachusetts, United States of America.
PLoS One. 2015 Apr 8;10(4):e0122044. doi: 10.1371/journal.pone.0122044. eCollection 2015.
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.
研究胰岛细胞对葡萄糖刺激的反应对于理解健康和疾病状态下的细胞功能至关重要。大多数功能测定是在整个胰岛或细胞群体上进行的，导致在单细胞水平上观察结果平均化且信息丢失。我们展示了在完整胰岛的单个细胞中检测对多次葡萄糖刺激的钙流的方法。野生型小鼠胰岛主要包含对三次（共三次）连续高葡萄糖刺激有反应的细胞，而糖尿病胰岛（db/db或NOD）的细胞反应频率较低或根本无反应。对成像的胰岛也进行内分泌标志物免疫染色，以将单个细胞的钙流谱与基因表达相关联。强力产生钙流的野生型小鼠胰岛细胞表达β细胞标志物（胰岛素/ NKX6.1），而在低葡萄糖水平反向产生钙流的胰岛细胞表达α细胞标志物（胰高血糖素）。糖尿病小鼠胰岛显示出功能失调的β细胞比例较高，这些细胞对葡萄糖刺激反应不佳。β细胞中大多数钙内流反应失败发生在第二次和第三次高葡萄糖刺激时，强调了多次连续葡萄糖刺激对于评估胰岛细胞完整功能的重要性。对人胰岛细胞也进行了评估，结果显示存在功能性α细胞和β细胞。这种将胰岛对多次葡萄糖刺激的反应与基因表达测定相关联的分析方法扩展了对β细胞功能和疾病状态的理解。
