Reporter assays using the ؊2.0-kb promoter revealed that this region contains a functional PPAR␥-responsive element. Gel mobility shift and chromatin immunoprecipitation assays showed that endogenous PPAR␥ protein binds to the perilipin promoter. PPAR␥2, an isoform exclusively expressed in adipocytes, was found to be the most potent regulator from among the PPAR family members including PPAR␣ and PPAR␥1. These results make evident the fact that perilipin gene expression in differentiating adipocytes is crucially regulated by PPAR␥2, providing new insights into the adipogenic action of PPAR␥2 and adipose-specific gene expression, as well as potential anti-obesity pharmaceutical agents targeted to a reduction of the perilipin gene product.Adipocytes are the major reservoir of energy stored in the form of triacylglycerol in the body. Triacylglycerol is stored within intracellular lipid droplets covered by a monolayer of phospholipids, free cholesterol, and proteins. In times of energy need, for example, as brought about by activities such as fasting and/or exercise, catecholamines rapidly activate cAMP-dependent protein kinase (PKA), 1 resulting in hydrolysis of triacylglycerol as catalyzed by activated hormone-sensitive lipase.

Monoacylglycerol lipase (MAGL) is a major serine hydrolase that hydrolyzes 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain. Because 2-AG and AA are endogenous biologically active ligands in the brain, inhibition of MAGL is an attractive therapeutic target for CNS disorders, particularly neurodegenerative diseases. In this study, we report the structure-based drug design of novel piperazinyl pyrrolidin-2-ones starting from our hit compounds 2a and 2b. By enhancing the interaction of the piperazinyl pyrrolidin-2-one core and its substituents with the MAGL enzyme via design modifications, we identified a potent and reversible MAGL inhibitor, compound (R)-3t. Oral administration of compound (R)-3t to mice decreased AA levels and elevated 2-AG levels in the brain.

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SREBPs1 are synthesized as membrane-bound precursor proteins. As long as intracellular sterol concentrations are sufficient, SREBPs remain bound to the endoplasmic reticulum and the nuclear envelope. Upon sterol deprivation, the NH 2 -terminal portion containing the basic helix-loop-helix leucine zipper domain is cleaved by a two-step process that occurs mainly in the Golgi apparatus (1). The mature SREBPs are then transported into the nucleus in a ␤-importin-dependent manner (2) and stimulate the transcription of genes involved in cholesterol and fatty acid metabolism. The SREBP family comprises three subtypes: SREBP-1a and SREBP-1c, which are derived from a single gene by alternative promoters and splicing, and SREBP-2, which is derived from a different gene. In addition to the sterol regulatory cleavage system, rapid degradation by the ubiquitin-proteasome pathway and another posttranslational modification, sumoylation, also regulate SREBP activity in the nucleus (3, 4). All of the data reported so far support the notion that SREBP-1c and SREBP-2 mainly regulate the transcription of genes involved in fatty acid synthesis and cholesterol metabolism, respectively, whereas SREBP-1a regulates both (5-7). SREBP-1a is the more potent activator of transcription than SREBP-1c because of a more extensive transactivation region but is expressed at a much lower level than SREBP-1c in most organs.We have identified several SREBP-responsive genes by a PCR subtraction method using a Chinese hamster ovary (CHO) cell line (CHO-487) expressing a nuclear form of human SREBP-1a with a LacSwitch inducible mammalian expression system (8). The benefit of this cell line is that it affords a higher induction of target genes, which is a crucial feature so as not to overlook any candidates, rather than a CHO cell line expressing SREBP-1c, which does not afford such a high induction level. Furthermore, the inducible genes in this cell line likely include those that are SREBP-2-responsive. Through this method we have detected SREBP-responsive genes such as the 3-hydroxy-3-methylglutaryl (HMG) CoA synthase and the stearoyl CoA desaturase-1 genes and further the ATP-citrate lyase gene (8), which catalyzes acetyl CoA synthesis. We also detected insulin-inducing gene-1, which was recently demonstrated to be a novel SCAP-binding protein regulating the proteolytic activation of SREBPs (9, 10). In the course of screening for SREBP-responsive genes, we have cloned and characterized the 5Ј-flanking region of the human SI gene.Cholesterol biosynthesis in mammals requires more than 30 enzymes, and most of this enzymatic activity is under sterolmediated feedback control (11). In the later stage of this pathway, SI catalyzes the conversion of the 8-ene isomer into the 7-ene isomer. In addition to its role in sterol isomerization, SI also functions as a multidrug-binding protein for various drugs, including the Ca 2ϩ antagonist emopamil, the immunosuppressant SR31747A, and the antiestrogen tamoxifen (12-15). Furthermore, recent genetic analyses revea...

Sterol regulatory element-binding protein-1 (SREBP-1) has been thought to be a critical factor that assists adipogenesis. During adipogenesis SREBP-1 stimulates lipogenic gene expression, and peroxisome proliferator-activated receptor γ (PPARγ) enhances perilipin (plin) gene expression, resulting in generating lipid droplets (LDs) to store triacylglycerol (TAG) in adipocytes. Plin coats adipocyte LDs and protects them from lipolysis. Here we show in white adipose tissue (WAT) of plin−/− mice that nuclear active SREBP-1 and its target gene expression, but not nuclear SREBP-2, significantly decreased on attenuated LD formation. When plin−/− mouse embryonic fibroblasts (MEFs) differentiated into adipocytes, attenuated LDs were formed and nuclear SREBP-1 decreased, but enforced plin expression restored them to their original state. Since LDs are largely derived from the endoplasmic reticulum (ER), alterations in the ER cholesterol content were investigated during adipogenesis of 3T3-L1 cells. The ER cholesterol greatly reduced in differentiated adipocytes. The ER cholesterol level in plin−/− WAT was significantly higher than that of wild-type mice, suggesting that increased LD formation caused a change in ER environment along with a decrease in cholesterol. When GFP-SREBP-1 fusion proteins were exogenously expressed in 3T3-L1 cells, a mutant protein lacking the S1P cleavage site was poorly processed during adipogenesis, providing evidence of the increased canonical pathway for SREBP processing in which SREBP-1 is activated by two cleavage enzymes in the Golgi. Therefore, LD biogenesis may create the ER microenvironment favorable for SREBP-1 activation. We describe the novel interplay between LD formation and SREBP-1 activation through a positive feedback loop.