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Carbohydrate Response Element Binding Protein (ChREBP) is a glucose-responsive transcription factor that activates genes involved in de novo lipogenesis in mammals. However, the molecular mechanism involved in ChREBP activation has not been elucidated. The conserved N-terminal region of ChREBP contains putative Nuclear Localization (NLS) and Nuclear Export (NES) signals, suggesting that nucleocytoplasmic shuttling plays a role in glucose regulation. To analyze cellular localization of ChREBP as a function of glucose, we used confocal microscopy to observe FLAG-tagged ChREBP in INS-1 cells, a glucose-responsive rat beta cell line. ChREBP is predominantly cytoplasmic under low and high glucose conditions; however addition of a nuclear export inhibitor Leptomycin B resulted in nuclear trapping of ChREBP independent of glucose. To test whether nuclear localization is sufficient for ChREBP activation, we mutated conserved residues in and around the NES. Mutating ChREBP at two residues within the NES led to nuclear accumulation of ChREBP, but surprisingly this mutation also blocked ChREBP activation. Additional mutations in the NES region of ChREBP identified forms that lost NES function, but retained glucose regulation. We conclude that 1) nucleocytoplasmic shuttling is not essential for glucose regulation of ChREBP and 2) that an unknown nuclear glucose-dependent event is required for activation. [ABSTRACT FROM AUTHOR]