Rodent pancreatic islet biology in health and disease

2009年，我们进一步开发了多色流式细胞术和定量基因表达系统，以表征健康小鼠和正在发展中或最近患有自身免疫性糖尿病的小鼠的内分泌细胞数量和功能。具体观察结果： 1.使用β细胞DNA含量分析(通过标准的BrdU掺入技术验证)，我们发现与健康小鼠的β细胞相比，糖尿病发病时的β细胞增殖增加(至少5-6倍)。在糖尿病发病时观察到的β细胞增殖与严重胰岛素抵抗小鼠模型的结果一致，后者通常会随着时间的推移而出现显着的β细胞增殖。我们测试了自身免疫性糖尿病中的β细胞增殖是由高血糖还是胰岛炎症引起的，数据清楚地表明是前者。例如，糖尿病前期小鼠的增殖细胞频率明显低于最近患糖尿病的小鼠，尽管糖尿病前期小鼠的胰岛被严重渗透，并且发生了实质性的胰岛损伤。今年出版了一份报告这些观察结果的手稿。 2.利用流式细胞术和荧光免疫组织化学方法，我们测定了糖尿病形成过程中小鼠胰岛β细胞相对于α细胞或增量细胞的频率。除了预期的β细胞枯竭之外，我们还观察到了同样严重的阿尔法和德尔塔细胞的丧失。与小的、亚胰岛大小的内分泌细胞团相比，这种来自胰岛以外的非β细胞枯竭在较大的胰岛中更为普遍。此外，在自身免疫的β细胞破坏期间，胰岛的α细胞和三角洲细胞的增殖率并没有增加，这与β细胞不同。我们的数据表明，随着自身免疫性糖尿病的发展，内分泌细胞在较大的胰岛和较小的内分泌细胞群之间的分布发生变化，这可能在糖尿病受试者观察到的矛盾的激素反应中发挥作用。例如，虽然糖尿病患者通常表现出高于正常循环中的胰升糖素水平，但正常的胰升糖素对低血糖的反应通常是迟钝的。今年出版了一份报告这些观察结果的手稿。 3.我们开发了一种可以快速、准确地测量总胰腺内分泌细胞数量的技术。利用目前的技术，已有许多药物被报道增加“胰岛细胞团”，但这种评估通常是使用繁琐和不精确的免疫组织学技术得出的。利用我们正在开发的流式细胞仪系统，我们可以跟踪参与导致自身免疫性糖尿病的过程的β细胞数量的下降，以及据信发生在胰岛素抵抗状态或某些胰岛生长因子(例如，胰高血糖素样肽1受体激动剂)治疗后的β细胞数量的增加。此外，我们已经确定了测量单个胰岛细胞亚群中基因转录本的技术，报道这些观察结果的手稿还没有出版。

During 2009, we further developed multicolor flow cytometry and quantitative gene expression systems to characterize endocrine cell number and function in both healthy mice and mice developing or with recent autoimmune diabetes. Specific observations: 1. Using beta cell DNA content analysis (validated using standard BrdU incorporation techniques), we found increased (at least 5-6 fold) beta cell proliferation at diabetes onset compared to beta cells from healthy mice. The observed beta cell proliferation at diabetes onset is consistent with results from severe insulin-resistance mouse models which typically develop marked beta cell hyperplasia over time. We tested whether the beta cell proliferation in autoimmune diabetes is caused by hyperglycemia or the islet inflammation, with data clearly suggesting the former. For instance, proliferating cell frequency is markedly lower in prediabetic mice compared to recently diabetic animals, even though prediabetic mouse islets are severely infiltrated and substantial islet injury has occurred. A manuscript reporting these observations was published this year. 2. Using flow cytometry and fluorescence immunohistochemistry, we have determined the frequency of mouse pancreatic beta cells relative to alpha- or delta-cells during diabetes development. In addition to the expected beta cell depletion, we observed an equally severe loss of alpha and delta cells. This other-than-beta-cell depletion from islets was much more prevalent in larger islets compared with small, sub-islet-sized endocrine cell clusters. Also, during autoimmune beta cell destruction, islet alpha- and delta-cell proliferation rates did not increase, unlike beta cells. Our data suggest that the distribution of endocrine cells between larger islets and small endocrine cell clusters changes as autoimmune diabetes develops, and may play a role in the paradoxical hormonal responses observed in diabetic subjects. For instance, while patients with diabetes typically display higher than normal circulating glucagon levels, the normal glucagon response to hypoglycemia is typically blunted. A manuscript reporting these observations was published this year. 3. We have developed a technique that can quickly and with some precision measure total pancreatic endocrine cell numbers. Using current techniques, a number of agents have been reported to increase "islet cell mass", but such assessments are typically drawn using laborious and imprecise immunohistological techniques. With the flow cytometric systems we are developing, we can track both the decline in beta cell numbers involved in the processes leading to autoimmune diabetes, and the increase in beta cell numbers believed to occur in insulin resistant states or following treatment with certain islet growth factors (e.g. glucagon-like peptide 1 receptor agonists). Further, we have identified techniques for measuring the gene transcripts within individual islet cell subsets, and a manuscript reporting these observations is not "in press."