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.
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