The "Metabolic Syndrome" is characterized by a cluster of metabolic defects, including obesity, insulin resistance, and elevated plasma lipid levels. This syndrome predisposes individuals to develop Type 2 diabetes and cardiovascular disease. Although the relationship among these metabolic defects is not well understood, it has been shown that treatments which lower plasma lipid levels are beneficial for these patients. Our goal is to use enhancer and suppressor genetics to identify potential new therapeutic targets for regulation of lipid metabolism. The Sterol Regulatory Element Binding Proteins (SREBPs) are a family of bHLH transcription factors that control the expression of genes involved in lipid metabolism in mammals, including those encoding the enzymes of cholesterol and fatty acid biosynthesis and transport as well as adipocyte differentiation. The activity of these transcription factors is post-translationally regulated: the inactive form is a transmembrane protein that is localized to the ER and nuclear envelope and is activated by two proteolytic cleavages that release the amino-terminal bHLH domain to allow entry into the nucleus. This proteolysis is regulated by nutritional signals, including cellular sterol and unsaturated fatty acid levels. The transcript level of SREBP is also regulated by nutritional signals including insulin signaling. In C. elegans, there is a single SREBP homolog,
sbp-1 (Sterol Regulatory Element Binding Protein-1), encoded by Y47D3B.7. We generated a deletion in the gene by excision of a Tc1 element, resulting in removal of the C-terminal regulatory regions of the gene while leaving the amino-terminal bHLH transcription factor domain intact. This mutation is a temperature-sensitive loss-of-function, and results in embryonic or larval arrest and pale intestine (Pin) phenotypes, similar to the RNAi phenotype. A second, weaker allele results in a viable Pin phenotype. The Pin phenotype results from defects in lipid synthesis and storage: well-fed
sbp-1 mutants have a paucity of lipid droplets in the intestine, resembling the phenotype of starved worms. The intestines of these animals stain poorly with lipid dye Sudan black as compared to the wildtype, and lipid profiles of
sbp-1 (ts) mutants indicate defects in fatty acid and triacylglycerol synthesis. Furthermore, although C. elegans is auxotrophic for cholesterol, we have shown that
sbp-1 mutants are hypersensitive to cholesterol deprivation, perhaps due to the overall lipid deficiencies in these animals. Examination of an
sbp-1p::GFP reporter construct indicates that the
sbp-1 gene is expressed in the intestine from early embryogenesis through adulthood, consistent with the intestinal phenotype. Two of the proteins involved in regulated cleavage of SREBP in mammals, Site 2 Protease (S2P) and SREBP Cleavage Activating Protein (SCAP) also are conserved in worms. RNAi experiments with these genes indicate that they interact with each other to give strong Pin phenotypes, and also that RNAi of S2P can enhance the weak
sbp-1 allele to give a strong Pin phenotype. Therefore, we believe that aspects of regulation of SREBP cleavage are conserved in worms as well. Because dauer formation has an (opposite) effect on lipid accumulation in the intestine, genetic interactions were examined between
sbp-1 and dauer-constitutive mutations in the
daf-2 (insulin receptor) and
daf-7 (TGF-b) pathways.
sbp-1 mutants or RNAi are weakly suppressed for the Pin phenotype by mutations in both pathways.