Growth and development of islets and beta-cells in the pancreas

我们与雷恩实验室合作，模拟了CDK4对胰腺切除后β细胞增殖的影响。在糖尿病中，产生胰岛素的胰岛β细胞要么被破坏，要么严重耗尽，要么功能受损。因此，更换功能性的β细胞团将促进临床糖尿病的治疗。利用具有靶向细胞周期蛋白依赖性激酶4(CDK4)基因座修饰的小鼠，我们已经阐明了CDK4在调节β细胞质量中的重要性。CDK4基因缺陷的小鼠表现出-细胞发育不全并发展为糖尿病，而在表达组成活性的CDK4R24C激酶的小鼠中观察到β细胞的增殖。在这项研究中，我们使用部分胰腺切除(PX)模型研究了CDK4调节的控制β细胞再生的机制。为了准确地研究再生过程的动力学，我们进行了基于DNA类似物的谱系追踪研究，然后进行了数学建模。在PX后一周内，我们观察到胰岛β细胞和导管上皮细胞大量增殖。有趣的是，数学模型表明，CDK4R24C胰腺中促进细胞增殖的两种机制可以解释这些数据：(1)加速已进入周期的细胞的复制，(2)将静止的细胞重新招募到活跃的细胞周期中。此外，在PX术后24-48小时内，表达转录因子PDX-1的导管上皮细胞显著增加。我们还在导管上皮中检测到胰岛素阳性细胞，并在CDK4R24C胰腺中检测到显著增加的胰岛样细胞团。因此，我们认为CDK4不仅促进了β细胞的自我复制，而且促进了导管上皮中的β细胞前体细胞的激活。这些发现有力地表明，CDK4通过促进细胞周期进程和招募静止的细胞进入细胞周期来控制β细胞质量。因此，利用CDK4活性恢复β细胞团对糖尿病具有潜在的治疗重要性。 我们与HARA实验室合作，根据他们的数据对胰岛发育的各个方面进行了定量了解： (1)关于人类胰岛中不同激素分泌细胞类型(α、高血糖素、β、胰岛素、β、生长抑素)的组织结构的新报告强调了人和小鼠胰岛之间的显著差异，提出了对小鼠胰岛研究与人类胰岛生理学的相关性的质疑。在这里，我们研究了 人和老鼠的小岛的建筑。我们研究了不同小鼠模型的胰岛，包括ob/ob、db/db和怀孕。 老鼠。我们还检查了猴子、猪、兔子和鸟类的胰岛，以进行进一步的比较。尽管总体上存在差异 在这些物种中，它们的胰岛大小的分布密切重叠， 除了在鸟类的胰腺中，d细胞群占主导地位(包括单胞体和簇状)以及少量的 小岛的数量。在人和猴的胰岛中观察到明显较大的胰岛(&gt；10,000；m~2)，在人和猴的胰岛中也观察到明显的大小的胰岛。 OB和怀孕的小鼠。在所有物种中，胰岛内的α、β和三角洲细胞的比例在不同的胰岛之间存在差异。 检查过了。此外，a-细胞和d-细胞在同一物种中的分布也存在差异。总而言之，人类 老鼠的胰岛具有共同的结构特征，这可能反映了对胰岛素的需求。对胰岛结构的比较研究可能有助于更好地了解胰岛的发育和功能。 (2)追踪特定细胞群体在健康和疾病中的变化是生物医学研究的重要目标。准确监测胰腺β细胞的增殖和胰岛生长是一个具有挑战性的研究领域。我们开发了一种方法，用为ImageJ(rsb.info.nih.gov/ij/)编写的宏来捕获带有荧光标记的β细胞的转基因小鼠完整胰腺中的β细胞分布。总的胰岛面积、胰岛数量和大小分布根据每个胰岛和小群的胰岛的特定参数和位置来量化。胰岛的整个分布现在可以在三维上绘制出来，每个胰岛的大小和形状的分布信息允许一目了然地对整个β细胞面积的变化进行定量和定性的比较。 (3)胚胎和新生儿内分泌细胞连续增殖，形成分枝状索状结构。我们的研究揭示了新生儿胰腺中沿着大血管分布的长长的相互连接的胰岛。阿尔法细胞横跨细长的胰岛样结构，我们假设这些结构代表分裂的地点，并促进最终形成离散的胰岛。对胰岛大小分布的详细分析也支持胰岛分裂的发生。位于这些假定切割位点的阿尔法细胞同时表达前激素转换酶2和1/3(分别为PC2和PC1/3)，而成人的阿尔法细胞仅表达PC2。PC1/3在这些新生儿α细胞中的表达导致原胰高血糖素前体被加工成胰高血糖素样肽1(GLP-1)，从而导致这种重要的β细胞生长因子的局部产生。我们认为，胰岛的形成是由毗邻的内分泌细胞增殖后的分裂过程发生的，而不是通过分离的β细胞和胰岛的局部聚集或融合而发生的。

We have collaborated with the Rane laboratory in modeling the effects of CDK4 on beta-cell proliferation after a pancreatectomy. Insulin-producing pancreatic islet beta cells are either destroyed, severely depleted, or functionally impaired in diabetes. Therefore, replacing functional beta cell mass would advance clinical diabetes management. Using mice with targeted cyclin-dependent kinase 4 (Cdk4) loci modifications, we have illustrated the importance of Cdk4 in regulating beta cell mass. Cdk4-deficient mice display β-cell hypoplasia and develop diabetes, whereas beta cell hyperplasia is observed in mice expressing a constitutively active Cdk4R24C kinase. In this study, we examined the Cdk4-regulated mechanisms controlling beta cell regeneration using a partial pancreatectomy (PX) model. To investigate the kinetics of the regeneration process precisely, we performed DNA analog-based lineage-tracing studies followed by mathematical modeling. Within a week after PX, we observed considerable proliferation of islet beta cell and ductal epithelial cells. Interestingly, the mathematical model showed that two mechanisms of enhanced cell proliferation in Cdk4R24C pancreas could account for the data: (1) accelerated replication of cells already in cycle, and (2) recruitment of quiescent cells into the active cell cycle. Moreover, within 24-48 hours post-PX, ductal epithelial cells expressing the transcription factor Pdx-1 dramatically increased. We also detected insulin-positive cells in the ductal epithelium along with a significant increase of islet-like cell clusters in the Cdk4R24C pancreas. We thus conclude that Cdk4 not only promotes beta cell self-duplication, but also facilitates the activation of beta cell progenitors in the ductal epithelium. These findings strongly suggest that Cdk4 controls beta cell mass by both enhancing cell cycle progression and recruiting quiescent cells to enter the cell cycle. Harnessing Cdk4 activity to restore beta cell mass is therefore of potential therapeutic importance for diabetes. We have collaborated with the Hara laboratory in formulating a quantitative understanding of various aspects of islet development from their data: (1) Emerging reports on the organization of the different hormone-secreting cell types (alpha, glucagon; beta, insulin; and delta, somatostatin) in human islets have emphasized the distinct differences between human and mouse islets, raising questions about the relevance of studies of mouse islets to human islet physiology. Here, we examined the differences and similarities between the architecture of human and mouse islets. We studied islets from various mouse models including ob/ob and db/db and pregnant mice. We also examined the islets of monkeys, pigs, rabbits and birds for further comparisons. Despite differences in overall body and pancreas size as well as total b-cell mass among these species, the distribution of their islet sizes closely overlaps, except in the bird pancreas in which the d-cell population predominates (both in singlets and clusters) along with a small number of islets. Markedly large islets (>10,000 μm2) were observed in human and monkey islets as well as in islets from ob/ ob and pregnant mice. The fraction of alpha-, beta- and delta-cells within an islet varied between islets in all the species examined. Furthermore, there was variability in the distribution of a- and d-cells within the same species. In summary, human and mouse islets share common architectural features that may reflect demand for insulin. Comparative studies of islet architecture may lead to a better understanding of islet development and function. (2) Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. Precisely monitoring pancreatic beta-cell proliferation and islet growth is a challenging area of research. We have developed a method to capture the distribution of beta-cells in the intact pancreas of transgenic mice with fluorescence-tagged beta-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Total beta-cell area, islet number and size distribution are quantified with reference to specific parameters and location for each islet and for small clusters of beta-cells. The entire distribution of islets can now be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and qualitative comparison of changes in overall beta-cell area at a glance. (3) Endocrine cells proliferate contiguously, forming branched cord-like structures in both embryos and neonates. Our study has revealed long stretches of interconnected islets located along large blood vessels in the neonatal pancreas. Alpha-cells span the elongated islet-like structures, which we hypothesize represent sites of fission and facilitate the eventual formation of discrete islets. The occurrence of islet fission is also supported by a detailed analysis of the islet-size distribution. The alpha-cells at these putative cleavage sites express both prohormone convertase 2 and 1/3 (PC2 and PC1/3, respectively), whereas alpha-cells in the adult express only PC2. The expression of PC1/3 in these neonatal alpha-cells results in the processing of the proglucagon precursor into glucagon-like peptide 1 (GLP-1), thereby leading to local production of this important beta-cell growth factor. We propose that islet formation occurs by a process of fission following contiguous endocrine cell proliferation, rather than by local aggregation or fusion of isolated beta-cells and islets.