This proposal aims to functionally determine the roles that Tcf7l2 play on glucose metabolism. Genome-wide association studies (GWAS) have consistently implicated non-coding variation within the TCF7L2 locus with risk of type 2 diabetes (T2D). While this represents the strongest genetic determinant for T2D risk in humans, it remains unclear how these non-coding variants affect disease etiology. We hypothesize that these associated non-coding regions harbor variants that alter the activity of long-range cis-regulatory elements controlling TCF7L2 expression would mediate the increased risk to disease. Over the past 3 years the PIs on this application have generated extensive resources and data that support this idea. We show that genomic interval associated with T2D harbors long-range enhancers regulating the temporal- spatial expression patterns of TCF7L2, including expression in tissues with a role in glucose homeostasis. To further determine whether variation in Tcf7l2 expression results in altered glucose homeostasis, we developed a Tcf7l2 copy number allelic series in mice. We show that a null Tcf7l2 allele (knockout mice) leads, in a dose- dependent manner, to better glucose tolerance. Conversely, transgenic mice harboring extra Tcf7l2 copies (with increased expression of the gene) display opposite phenotypes, including glucose intolerance. We also show how the metabolic phenotypes in these mice are mediated primarily from extra-pancreatic tissues. These results directly demonstrate that Tcf7l2 is a key regulator of glucose tolerance, and highlight its roles in peripheral tissues as mediators of T2D risk in humans. In this application, we propose to capitalize on the unique molecular and live reagents that we have developed and address outstanding questions using genomics, in vitro cellular-based and in vivo mouse-based approaches: (i) What is the mechanism by which Tcf7l2 regulates glucose tolerance in vivo?, (ii) Which tissues participate in the Tcf7l2-mediate regulation of glucose tolerance?, (iii) What are the molecular downstream targets and pathways regulated by Tcf7l2 in these tissues? To address these questions we propose 2 aims. In Aim 1 we will carry out a detailed physiological, molecular and morphometric evaluation in our Tcf7l2 copy number mouse panel. We will also test the hypothesis that different splice isoforms of Tcf7l2 may result in glucose metabolism phenotypes. In Aim 2, we will establish the tissues that play a role in Tcf7l2- mediated control of glucose metabolism. We will investigate the putative physiological roles of Tcf7l2 in brain/hypothalamus, adipose tissue, and liver by tissue-specific ablations of this gene. The results of these studies will significantly impact our understanding of T2D pathogenesis, provide a new paradigm for the experimental follow-up of GWAS with noncoding variation, and identify new therapeutic pathways in tissues with yet unknown Tcf7l2-mediated functions.