Abstract Evolutionary pressure has favored mechanisms that allow the body to efficiently store nutrients as fat when food is abundant as a safeguard against occasional famine. With the dramatic changes in modern lifestyle including consumption of high carbohydrate and high fat foods, these mechanisms may now be contributing to a major epidemic of obesity in the US where the majority of the population is overweight. Glucose is not only a major fuel of all mammalian tissues but also a source of carbon for fat and protein synthesis. The liver is the principal organ responsible for the conversion of excess dietary carbohydrate into triglycerides. Ingestion of a high carbohydrate diet induces transcription of more than 20 genes involved in the conversion of glucose to storage fat. A transcription factor with specificity for carbohydrate responsive elements (ChRE) found in the promoters of multiple genes required for lipogenesis and which displayed appropriate dietary responsive regulation, was first identified in my laboratory following purification from rat liver and termed carbohydrate response element binding protein, ChREBP. The complete process by which ChREBP is activated in response to excess carbohydrate in order to induce the transcription of lipogenesis enzyme genes and then is turned off is not yet fully understood. We have shown that glucose and cAMP have antagonistic roles in the regulation of ChREBP activities in part through phosphorylation and dephosphorylation of multiple sites on ChREBP. Glucose stimulates dephosphorylation of at least some of these sites by activating a Xu5P-stimulated protein phosphatase, Xu5P-PP2A. More recently we have found that the interaction of ChREBP with 14-3-3 is one of the most important steps in regulation of the nuclear localization of ChREBP. The phosphorylation of ChREBP enhances the binding to 14-3-3 and is essential for export of ChREBP out of the nucleus under fasting conditions. In addition, we found that certain metabolites in fasted liver, but not in fed liver, promote the interaction of ChREBP with 14-3-3, and thereby export of ChREBP out of the nucleus. We have identified the metabolites as ketone bodies. To characterize the metabolites further we propose to (1) determine the biochemical mechanism by which they promote the interaction between ChREBP and 14-3-3, (2) determine their physiological significance in inhibition of ChREBP activities in hepatocytes, and (3) investigate whether the metabolites are signaling compounds for regulation of the subcellular localization of ChREBP in response to nutrients and fasting in vivo. We also will investigate other glucose signaling compounds in high sucrose fed rat liver extracts that may increase importin binding and/or destabilize 14-3-3 binding to promote the nuclear import of ChREBP. X-ray crystallography will be used to investigate the mechanisms of metabolite regulation of ChREBP binding to 14-3-3 and importin at an atomic level.