PROJECT SUMMARY Insulin resistance, excess hepatic glucose production, and impaired insulin secretion are the hallmarks of type 2 diabetes mellitus (T2DM), and tissue iron levels significantly affect all three. In mice and humans, we have shown that high iron impairs insulin secretion and down regulates leptin and adiponectin. Our preliminary data show further that these effects of iron are fuel-dependent, with much of this difference based on higher iron levels supporting higher levels of fat oxidation. Our mechanistic work on these effects of iron has revealed the involvement of numerous pathways, including transcriptional regulation (notably by CREB, FoxO1, and PGC1α) and nutrient/metabolite signaling (AMPK, sirtuins, and mTOR). Thus, the effects of iron are complex, pleiotropic, and cannot be explained by invoking a single linear signal transduction pathway. Recently our work on the mechanism by which iron regulates leptin secretion has revealed a unifying concept for these pleiotropic effects: High tissue iron down-regulates a central integrator of nutrient and redox status, the O-linked N-acetyl glucosamine (O-GlcNAc) pathway. This pathway results in the O-GlcNAc modification of most transcription factors and numerous enzymes that regulate metabolism. Activation of the pathway is often a direct readout of cellular nutrient fluxes, and we have shown it to be sufficient to induce changes in insulin sensitivity, insulin secretion, and hepatic glucose metabolism in ways that recapitulate T2DM. A second pathway that responds to both nutrient and oxidative stresses is the hypoxia-sensing pathway. Like the O-GlcNAc pathway, it functions at both ends of two metabolic spectra—low glucose and low oxygen as well as high glucose and oxidative stress. The pathways regulate one another and interact in determining hepatic glucose production, insulin sensitivity, and insulin secretion. Importantly, both the O- GlcNAc and hypoxia pathways are not only relevant to pathologic iron overload and hypoxia, but regulate metabolism in normal physiology, across the very broad range of “normal” iron and in individuals at sea level. In sum, the O-GlcNAc and hypoxia pathways cooperate to sense the availability or excess of two essential elements required for oxidative metabolism, iron and oxygen. Based on the above, our published work, and Preliminary Data, we therefore hypothesize that these two pathways integrate these signals to regulate several metabolic pathways involved in the pathogenesis of T2DM. Modulation of the O-GlcNAc pathway by iron affects numerous signal transduction pathways, leading to broad-based changes in metabolism that globally alter fuel utilization to confer adaptive responses to either a lack or excess of iron. In parallel, the hypoxia pathway performs a parallel function based on oxygen availability or excess oxidant stress. Crosstalk between the two pathways can amplify their effects, resulting in integration and a “fine-tuning” of metabo...