RNAi screens reveal novel metabolic regulators : RIP140, MAP4k4 and the lipid droplet associated fat specific protein (FSP)27

Type 2 diabetes is associated with obesity and with the inability of pancreatic beta cells to secrete sufficient insulin to maintain normal blood glucose levels (Ahren 2005, Kahn et al. 2006, Marchetti et al. 2006, Salsali & Nathan 2006). However, mild forms of type 2 diabetes in obese patients are often associated with circulating insulin concentrations that are actually higher than observed in normal subjects. This paradox was resolved by the finding that peripheral tissues such as muscle and liver in such type 2 diabetic subjects are relatively resistant to the actions of insulin, prompting beta cells to secrete higher than normal levels of insulin. Insulin resistance in muscle of obese subjects, in particular, usually appears prior to the ultimate failure of beta cells (Petersen & Shulman 2002, Ahren 2005). This insulin resistance in skeletal muscle in turn is thought to result from defects in the signalling pathways whereby insulin enhances translocation of intracellular glucose transporters (GLUT4) to the cell surface membrane, while the total expression of the GLUT4 protein is the same in normal and diabetic subjects (Huang & Czech 2007). Interestingly, such syndromes as obesity and lipdystrophy are thought to confer insulin resistance to skeletal muscle, even in subjects who are not diabetic (Sovik et al. 1996). When the beta cells of such subjects exhibit impaired insulin secretion, type 2 diabetes results. Thus, understanding the molecular connections between obese or lipodystrophic conditions and the induction of impaired insulin signalling in skeletal muscle is a major goal in the field. Over the past several years it has become apparent that adipose tissue in humans, mice and rats plays a key role in regulating insulin signalling in skeletal muscle. The basis for this conclusion includes experiments showing that adipose-specific deletions in key genes lead to systemic glucose intolerance and insulin resistance. For example, conditional ablation of GLUT4 in adipose tissue in mice causes impaired insulin signalling in skeletal muscle, glucose intolerance and even diabetes in some of the animals (Li et al. 2000, Rossetti et al. 1997, Stenbit et al. 1997). Conversely, selective expression of GLUT4 (Shepherd et al. 1993, Tozzo et al. 1995) at high levels in adipose tissue greatly improves insulin sensitivity in mice. Knockout of the gene encoding the adipose-specific protein adiponectin also leads to decreased insulin sensitivity in muscle and impaired glucose tolerance in vivo (Kubota et al. 2002, Pajvani & Scherer 2003, Haluzik et al. 2004, Civitarese et al. 2006), although one study failed to observe this effect (Ma et al. 2002). It has also been noted that adipose tissue-specific deficiency of peroxisome proliferator-activated receptor-γ (PPAR-γ) protects against high fat diet-induced insulin resistance (Jones et al. 2005). Furthermore, transplantation of adipose tissue from normal mice into lipodystrophic mice prevents the muscle insulin resistance and glucose intolerance of this condition (Gavrilova et al. 2000). Taken together, these and other data provide compelling evidence in support of the concept that adipose tissue metabolism strongly influences whole body glucose tolerance and skeletal muscle insulin sensitivity. Based upon the above considerations, we reasoned that adipose tissue was a suitable target tissue to search for novel proteins that regulate adipocyte metabolism directly and perhaps whole body metabolism indirectly. Identification of such proteins might provide useful insight into the molecular basis of insulin signalling or other aspects of adipocyte function, and may lead to potent targets for therapeutic agents. We thus developed a strategy for such identifications based upon a two step process (Powelka et al. 2006, Tang et al. 2006): (1) Selection of candidate genes/proteins from the entire database of mouse and human genes, based on high, selective expression in adipocytes and their regulation by changes in metabolic states, and (2) Screening the function of these candidate genes by RNA interference (RNAi)-based gene silencing in cultured adipocytes, using insulin signalling to glucose transport as a reporter assay. We developed methods for silencing genes with pools of short interfering RNA (siRNA) oligonucleotides in cultured 3T3-L1 adipocytes (Jiang et al. 2003) as well as in primary mouse and human adipocytes (Puri et al. 2007). Following successful functional analysis of several candidate genes with this method (Jiang et al. 2003, Mitra et al. 2004, Zhou et al. 2004, Powelka et al. 2006, Tang et al. 2006), we were able to miniaturize the procedure and perform gene silencing assays in a higher throughput mode (30–50 genes per week). These experiments have led to the identification of several genes that encode proteins with novel functions in regulating adipocyte biology. Three of these proteins we have discovered by this approach are briefly summarized in this review, and may be potential targets for development of therapeutic agents in future studies.