This flux rose to 1.3 mmol/g-DNA/2-days with the long-term FCCP treatment. UCP1 Ginsenoside-Re expression increased proline production by 127% compared to the pRev control. A previous study on the metabolic impact of forced UCP1 expression in 3T3-L1 adipocytes indicated a shift of glucose-derived carbon flux into lactate production instead of lipid synthesis. In this study, we mimicked the mitochondrial uncoupling effects of forced UCP1 expression by subjecting cultured 3T3-L1 to mild, but prolonged FCCP treatment. Similar to UCP1 expression, this treatment did not affect the transcript levels of PPARc or GPDH, two markers of adipocyte differentiation. Chemical uncoupling also reduced the accumulation of TG while increasing glucose uptake and lactate output. Model calculations revealed generally consistent metabolic flux changes for both FCCP and UCP1-mediated uncoupling, suggesting a common mechanism. Mitochondrial uncoupling through UCP1 expression and FCCP treatment both down-regulated de novo fatty acid synthesis, TG synthesis and storage, and lipolysis. The uncoupling also reduced the fluxes through several reactions in other parts of intermediary metabolism that directly or indirectly couple to fatty acid synthesis. These reactions included steps in the PPP, PEPCK-catalyzed formation of PEP, and citrate lyase catalyzed formation of cytosolic acetyl-CoA. In adipocytes, the PPP is a quantitatively important source of NADPH for de novo fatty acid synthesis. Not surprisingly, the activity of the PPP depends on the cellular demand for NADPH. This regulatory relationship suggests that the decrease in the PPP flux also reflects a metabolic adjustment to the uncoupling. Similarly, the reduced demand for fatty acid and TG Sophocarpine building blocks, respectively, could explain the reduced flux of acetyl-CoA and PEP. The estimated flux changes were generally consistent with the results of a recent study characterizing the impact of chemical uncoupling on gene expression in mature adipocytes.Expression profiling following short-term FCCP treatment found decreased transcript levels of a number of lipogenic enzymes, including CL, PEPCK, mitochondrial glycerol-3-phosphate acyltransferase, dihydroxyacetone-phosphate acetyltransferase, fatty acid synthase, HSL and ATGL.