This is a request for renewal of NIH grant DK31036 which was granted MERIT status at the last competitive renewal. This grant is now completing 33 years. Since its inception, the grant has been focused on the insulin receptor, and to a lesser extent IGF-1 receptor, their signaling capabilities and their alterations in physiologic and pathologic states. Over the course of this grant, we have made very substantial progress in understanding the signaling and biological roles of these receptors through both in vitro and in vivo studies. We have been pioneering in the use of homologous recombination gene targeting to study the role of the insulin receptor in insulin action in vivo through the creation of single knockouts of the insulin and IGF-1 receptors; combinatorial knockouts as polygenic models of Type 2 diabetes; and tissue-specific knockouts using the Crelox approach to determine the role of the insulin/IGF-1 receptors in individual tissues in normal physiology and the pathophysiology of diabetes and metabolic syndrome. In addition, we have performed a series of in vitro studies focusing on differential signaling by the insulin and IGF-1 receptors, as well as defining the actions of insulin and IGF-1 in different cell types. As a result, since the last renewal, this grant has provided support for a total of 44 published papers (24 original articles, 13 collaborative articles and 7 reviews) listed as references 1-44 in the Literature Cited Section of this grant. This work has led to development of new hypotheses about the nature of the insulin/IGF-1 signaling network, the contribution of insulin resistance in different organs to diabetes and the metabolic syndrome, as well as evidence on how tissues communicate with one another in insulin resistant states. Over the past 4 years, we have accomplished most of the goals set forth in the last renewal. In addition we have made a number of novel observations and developed new tools, including new data on the role of insulin action in the gut, the role of IR in IPS cells and the role of the unoccupied IR and IGFIR in control of imprinted gene expression. During the coming grant period we hope to continue the work begun during the current grant period, expand our understanding of insulin/IGFI action in vivo using new knockout models, complete development of new knockin mouse models for insulin/IGF-1 action and define the nature of unoccupied receptor signaling by identifying IR and IGFIR interacting proteins. While this is ambitious, based on our track record and the fact that many of the systems are in place and inter-related, we believe this can be done over the next 5 years.
The specific aims are to: 1. Further define the role of insulin signaling in the brain by extending studies on the interaction between insulin action in brain, aging, and control of brain function. This will include determining the role of insulin signaling in glial and neuronal cells through cell specific IR knockouts; determining the role of cholesterol metabolism in the brain on regulation of neural and metabolic function; and determining how altered insulin action in brain might alter metabolite distribution using high resolution spatial mass spectrometry. 2. Expand studies of the role of insulin and IGF-1 signaling in peripheral tissues using three new experimental paradigms: the inducible adipose specific 1R/IGF1R knockout to create inducible and remitting insulin resistance; the intestinal epithelial IR/IGF1R knockout to explore the role of insulin signaling in the gut and its interaction with the gut microbiome; and the use of human IPS cells before and after differentiation to muscle to study the role nature of insulin resistance in human cells ex vivo. 3. Determine the nature of signaling by the unoccupied insulin and IGF-1 receptors involved in control of gene expression from imprinted genes and miRNAs and apoptosis by analysis of insulin/IGFI R receptor mutants to define the structural determinants of these effects and through the use of mass spectroscopy based proteomics to identify receptor interacting proteins responsible for these actions. 4. Define the differential nature of insulin and IGF-1 receptor signaling in vivo by creation of a new generation of mouse models in which the insulin receptor is replaced by knockin of the IGF1 receptor or a chimeric insulin/IGF-1 receptor containing the extracellular domain of one receptor and the intracellular domain of the other to produce mice and cell lines that express each receptor or chimeric receptor in the tissue and temporal pattern of the other receptor to determine the in vivo nature of hormone specificity.
This aim was proposed in the previous renewal of the grant and the in vitro aspects have been largely completed, but the in vivo knockin studies have just begun with creation of our first knockin mouse.
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