Glucose effects on cellular functions such as gene expression require, in general, glucose metabolism at least to glucose-6-phosphate (G-6-P). However, the example of thioredoxin-interacting protein (TXNIP), a glucose-regulated gene involved in the cellular redox state and pancreatic beta cell apoptosis, demonstrates that this rule may not always apply. We found that aside form glucose, the nonmetabolizable sugars 2-deoxyglucose, which is still converted to G-6-P as well as 3-O-methylglucose (3-MG), which cannot be phosphorylated by glucokinase, stimulate TXNIP expression. In contrast, incubation of INS-1 beta cells with equimolar amounts (25 mM) of 1-glucose or mannitol had no effect on TXNIP expression as measured by real-time RT-PCR, eliminating the possibility of an osmotic effect. Also, glucose uptake into the cell is critical because phloretin, an inhibitor of glucose transporter 2, blunted the glucose effects. Moreover, the 3-MG effect was not restricted to a cell line and was observed in 293 cells and primary human islets. Incubation of INS-1 cells with 30mM mannoheptulose, an inhibitor of glucose metabolism, blunted all glucose-induced gene expression but left the 3-MG effects unaltered. Using transient transfection studies and deletion constructs of the human TXNIP promoter, we found that the effects of glucose and 3-MG were dependent on the same region of the TXNIP promoter containing an E-box repeat carbohydrate response element (ChoRE). Thus, these findings provide the first evidence for regulation of gene expression by 3-MG, which is independent of glucose metabolism and suggest that glucose and 3-MG regulate transcription by two distinct pathways converging at a common ChoRE. © 2006 American Chemical Society.