Studying lipolysis in adipocytes by combining siRNA knockdown and adenovirus-mediated overexpression approaches.

3T3-L1 adipocytes are widely used as a model system for studying hormone-stimulated lipolysis. However, these cells were limited in their utility for gain- and loss-of-function studies due to the low efficiency of their transfection with plasmid DNA or small interfering RNA (siRNA) oligos. In this chapter, we provide a review of two methods established for manipulation of protein expression in differentiated mature adipocytes. The use of electroporation allows a high-efficiency delivery of siRNA oligos and subsequent knockdown of specific gene expression. A centrifugation-assisted infection with recombinant adenovirus, on the other hand, enables robust overexpression of ectopic proteins. Most importantly, by combining siRNA electroporation with adenovirus infection, simultaneous manipulation of levels of two different proteins can be achieved in differentiated adipocytes. Through subsequent analyses of lipase activity in cell extracts and fatty acid or glycerol release from living cells, mutual interdependence between the two proteins in the context of basal and hormone-stimulated adipocyte lipolysis can be evaluated.