Fructose 2,6-bisphosphate (Fru-2,6-P-2) is an important metabolite that controls glycolytic and gluconeogenic pathways in several cell types. Its synthesis and degradation are catalyzed by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2). Four genes, designated Pfkfb1-4, codify the different PFK-2 isozymes. The Pfkfb3 gene product, ubiquitous PFK-2 (uPFK-2), has the highest kinase/bisphosphatase activity ratio and is associated with proliferation and tumor metabolism. A transgenic mouse model that overexpresses uPFK-2 under the control of the phosphoenolpyruvate carboxykinase promoter was designed to promote sustained and elevated Fru-2,6-P-2 levels in the liver. Our results demonstrate that in diet-induced obesity, high Fru-2,6-P-2 levels in transgenic livers caused changes in hepatic gene expression profiles for key gluconeogenic and lipogenic enzymes, as well as an accumulation of lipids in periportal cells, and weight gain. (c) 2007 Elsevier Inc. All rights reserved.