An excessive increase in body fat (white adipose tissue, WAT) mass, or adiposity, is the principal driver of obesity, and a risk factor for many diseases, including type 2 diabetes. Enlarged (hypertrophic) white adipocytes found in obese individuals release free fatty acids and secrete pro-inflammatory cytokines that contribute to β-cell dysfunction and systemic insulin resistance. Controlling adiposity by targeted modulation of adipocyte enzymes could offer an attractive molecular therapeutic-based alternative to current dietary approaches.

In prior work, we had developed an optimization-based method for metabolic flux analysis (MFA) that significantly reduced the required number of external flux measurements. Application of this MFA method to hypertrophic white adipocytes resolved the flux distribution through several cyclic pathways of lipid metabolism, and pointed to fatty acid oxidation and synthesis as key steps in determining the extent of de novo adipogenesis and subsequent lipid loading. In this study, we engineer a control site for exogenous modulation of intracellular fatty acid levels and characterize the metabolic and gene expression profiles of the modified white adipocytes. To exogenously modulate intracellular fatty acid levels, we forced the expression of a brown adipocyte respiratory uncoupling protein (UCP1) under the control of a tetracycline repressed promoter. In brown adipocytes, which are present in rodents but not in adult humans, UCP1 promotes cellular energy dissipation as heat.

After stable integration, the ucp1 gene product was continuously expressed, and reduced the total lipid accumulation by ca. 30 % without affecting several other adipocyte markers, such as cytosolic glycerol-3-phosphate dehydrogenase activity and leptin production. The expression of UCP1 also decreased glycerol output and increased glucose uptake, lactate output, and the sensitivity of cellular energy charge to nutrient removal. We further compared the engineered and wild-type adipocytes through MFA and gene expression profiling. MFA of the engineered white adipocytes indicated that the reduction in intracellular lipid through UCP1 reflected a down-regulation of fat synthesis, rather than an up-regulation of fatty acid oxidation. These findings were consistent with the results of the gene chip expression profiling analysis. Interestingly, the gene chip analysis revealed that very large numbers of genes (552 and 851, respectively) were significantly up- and down-regulated in the UCP1-expressing adipocytes. Taken together, our results describe a direct mechanism for lipid reduction in adipocytes by UCP1 and point to this protein as a potential target for obesity drug development. Future studies will examine the impact of additional modifications to UCP1-responsive pathways identified in this study, such as the pentose phosphate pathway and malate cycle.