Adaptation to environmental stress is a fundamental cellular process that promotes the maintenance of the physiologic steady state. Stress responses have been shown to induce numerous changes, such as activation of gene expression programs which have evolved to allow for the cell to promote its own survival. Many insights into the functional role of microRNAs (miRNAs) have recently been made, improving our understanding of how these small RNAs integrate into the already complex landscape of gene expression. Most notably, based on a number of miRNA knockout mouse models showing subtle phenotypes under steady-state conditions, a hypothesis has emerged suggesting the miRNA pathway contributes to cellular stress responses. In addition, the profiling of microRNAs from numerous disease states such as cancer, insulin resistance, and inflammation has identified dramatic changes in many abundant small RNA sequences indicating this pathway plays a significant role in adapting cells to changes in their metabolic environment.
While the endocrine pancreas is known to contribute to a multitude of diverse physiologic pathways, our understanding of the extent of its influence and the precise mechanisms dictating its growth and function remains incomplete. Our work on miRNAs in the pancreatic b-cell began with the profiling of small RNAs and the identification of miR-375, the most abundant microRNA in this cell type which is broadly conserved from human to zebrafish. Generation of the knockout mouse showed loss of miR-375 resulted in reduced β-cell proliferation in response to insulin resistance indicating the microRNA pathway mediates the compensatory expansion of this cell type. In light of the absence of any dramatic effect in the development of the islet or specification of the different endocrine cell populations in the miR-375 knockout mouse, these results began to suggest a larger role for this miRNA and its direct targets during stress responses. Our current research focus has been to continue to improve our understanding of the functional role of the microRNA pathway in the pancreatic β-cell. Based on the observations made in the miR-375 knockout mouse, the research goals of our group are:
- Identify additional microRNAs contributing to the growth and function of the β-cell during insulin resistance and characterize their role in compensatory mechanisms of the β-cell.
- Characterize the functional role of the direct targets of miR-375 and establish their mechanism of action in the β-cell.
Small RNA profiling has identified the silencing of miR-184 in the pancreatic islets of leptin-deficient ob/ob mice. In addition, the analysis of both loss and gain of function mouse lines established miR-184 as a negative regulator of β-cell secretion and proliferation by targeting two genes, the glutamate carrier Slc25a22, and Argonaute2 (Ago2), a key mediator of the miRNA pathway. Moreover, conditional deletion and transgenic over-expression both confirmed Ago2 necessary for the maintenance of b-cell mass and loss of Ago2 resulted in increased expression of several targets of miR-375 including Cadm1. In line with previous studies supporting its role as a growth suppressor, genetic deletion of Cadm1 resulted in increased β-cell mass indicating this target contributes to the regulatory role of miR-375 in this cell type. Lastly, administration of a ketogenic diet to ob/ob mice rescued `insulin sensitivity and the expression of miR-184, and in turn restored b-cell mass and Slc25a22 and Ago2 expression. In addition to establishing an integral role for the miRNA pathway in compensatory proliferation, these results illustrated the dynamic nature of miRNA function according to changes in insulin sensitivity. Our work has also implemented a proteomic approach to analyze the role of miR-375 and its targets on b-cell secretion. Here we showed many of the direct targets of this miRNA including Cadm1, Gphn, HuD/Elavl4 and Rasd1 all contribute to exocytosis in this cell type. Together, these results showed that numerous targets of independent miRNAs coordinately participate in both growth and function of the β-cell.
Our observations functionally associating miR-184 together with Ago2 and miR-375 now identify a network within the miRNA pathway that coordinately regulates the compensatory proliferation of the b-cell. Based on these studies, the future Aims of my group will continue to emphasize the study of genes pertinent to the pathogenesis of metabolic disease. To date, since identifying miR-375 in the pancreatic islet, our observations continue to reinforce the role of microRNAs in stress responses. Our future goals are to identify and characterize the mechanisms which regulate the microRNA pathway during changes in nutrient availability and how the regulation of this pathway ultimately contributes to the growth and function of the β-cell.