PPAR␥ is a member of the PPAR subfamily of nuclear receptors. In this work, the structure of the human PPAR␥ cDNA and gene was determined, and its promoters and tissue-specific expression were functionally characterized. Similar to the mouse, two PPAR isoforms, PPAR␥1 and PPAR␥2, were detected in man. The relative expression of human PPAR␥ was studied by a newly developed and sensitive reverse transcriptasecompetitive polymerase chain reaction method, which allowed us to distinguish between PPAR␥1 and ␥2 mRNA. In all tissues analyzed, PPAR␥2 was much less abundant than PPAR␥1. Adipose tissue and large intestine have the highest levels of PPAR␥ mRNA; kidney, liver, and small intestine have intermediate levels; whereas PPAR␥ is barely detectable in muscle. This high level expression of PPAR␥ in colon warrants further study in view of the well established role of fatty acid and arachidonic acid derivatives in colonic disease. Similarly as mouse PPAR␥s, the human PPAR␥s are activated by thiazolidinediones and prostaglandin J and bind with high affinity to a PPRE. The human PPAR␥ gene has nine exons and extends over more than 100 kilobases of genomic DNA. Alternate transcription start sites and alternate splicing generate the PPAR␥1 and PPAR␥2 mRNAs, which differ at their 5 -ends. PPAR␥1 is encoded by eight exons, and PPAR␥2 is encoded by seven exons. The 5 -untranslated sequence of PPAR␥1 is comprised of exons A1 and A2, whereas that of PPAR␥2 plus the additional PPAR␥2-specific N-terminal amino acids are encoded by exon B, located between exons A2 and A1. The remaining six exons, termed 1 to 6, are common to the PPAR␥1 and ␥2. Knowledge of the gene structure will allow screening for PPAR␥ mutations in humans with metabolic disorders, whereas knowledge of its expression pattern and factors regulating its expression could be of major importance in understanding its biology.White adipose tissue is composed of adipocytes, which play a central role in lipid homeostasis and the maintenance of energy balance in vertebrates. These cells store energy in the form of triglycerides during periods of nutritional affluence and release it in the form of free fatty acids at times of nutritional deprivation. Excess of white adipose tissue leads to obesity (1-3), whereas its absence is associated with lipodystrophic syndromes (4). In contrast to the development of brown adipose tissue, which mainly takes place before birth, the development of white adipose tissue is the result of a continuous differentiation/development process throughout life (2, 5). During development, cells that are pluripotent become increasingly restricted to specific differentiation pathways. Adipocyte differentiation results from coordinate changes in the expression of several proteins, which are mostly involved in lipid storage and metabolism, that give rise to the characteristic adipocyte phenotype. The changes in expression of these specialized proteins are mainly the result of alterations in the transcription rates of their genes.Several transcription fac...

The biochemical analysis of cellular trans-activators involved in promoter recognition provides an important step toward understanding the mechanisms of gene expression in animal cells. The promoter selective transcription factor, Sp1, has been purified from human cells to more than 95 percent homogeneity by sequence-specific DNA affinity chromatography. Isolation and renaturation of proteins purified from sodium dodecyl sulfate polyacrylamide gels allowed the identification of two polypeptides (105 and 95 kilodaltons) as those responsible for recognizing and interacting specifically with the GC-box promoter elements characteristic of Sp1 binding sites.

Increased activity of lipoprotein lipase (LPL) may explain the hypotriglyceridemic effects of fibrates, thiazolidinediones and fatty acids, which are known activators (and/or ligands) of the various peroxisome proliferator‐activated receptors (PPARs). Treatment with compounds which activate preferentially PPARalpha, such as fenofibrate, induced LPL expression exclusively in rat liver. In contrast, the antidiabetic thiazolidinedione BRL 49653, a high affinity ligand for PPARgamma, had no effect on liver, but induced LPL expression in rat adipose tissue. In the hepatocyte cell line AML‐12, fenofibric acid, but not BRL 49653, induced LPL mRNA, whereas in 3T3‐L1 preadipocytes, the PPARgamma ligand induced LPL mRNA levels much quicker and to a higher extent than fenofibric acid. In both the in vivo and in vitro studies, inducibility by either PPARalpha or gamma activators, correlated with the tissue distribution of the respective PPARs: an adipocyte‐restricted expression of PPARgamma, whereas PPARalpha was expressed predominantly in liver. A sequence element was identified in the human LPL promoter that mediates the functional responsiveness to fibrates and thiazolidinediones. Methylation interference and gel retardation assays demonstrated that a PPARalpha or gamma and the 9‐cis retinoic acid receptor (RXR) heterodimers bind to this sequence −169 TGCCCTTTCCCCC −157. These data provide evidence that transcriptional activation of the LPL gene by fibrates and thiazolidinediones is mediated by PPAR‐RXR heterodimers and contributes significantly to their hypotriglyceridemic effects in vivo. Whereas thiazolidinediones predominantly affect adipocyte LPL production through activation of PPARgamma, fibrates exert their effects mainly in the liver via activation of PPARalpha.

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We report the cDNA cloning of SREBP-2, the second member of a family of basic-helix4oop-helix4leucine zipper (bHLH-Zip) transcription factors that recognize sterol regulatory element 1 (SRE-1). SRE-1, a conditional enhancer in the promoters for the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A synthase genes, increases transcription in the absence of sterols and is inactivated when sterols accumulate. Human SREBP-2 contains 1141 amino acids and is 47% identical to human SREBP-la, the first recognized member ofthis family. The resemblance includes an acidic NH2 terminus, a highly conserved bHLH-Zip motif (71% identical), and an unusually long extension of 740 amino acids on the COOH-terminal side of the bHLH-Zip region. SREBP-2 possesses one feature lacking in SREBP-la-namely, a glutamine-rich region (27% glutamine over 121 residues). In vitro SREBP-2 bound SRE-1 with the same specificity as SREBP-la.In vivo it mimicked SREBP-la in activating transcription of reporter genes containing SRE-1. As with SREBP-la, activation by SREBP-2 occurred in the absence and presence of sterols, abolishing regulation. Cotransfection oflow amounts of pSREBP-la and pSREBP-2 into human embryonic kidney 293 cells stimulated transcription of promoters containing SRE-1 in an additive fashion. At high levels transcription reached a maximum, and the effects were no longer additive. The reason for the existence of two SREBPs and the mechanism by which they are regulated by sterols remain to be determined.A 10-base-pair (bp) element in the 5' flanking region confers sterol sensitivity upon the genes encoding the low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) synthase. This element, sterol regulatory element 1 (SRE-1), enhances transcription when cells are depleted of sterols, and loses its activity when sterols accumulate (1, 2). This feedback mechanism allows cells to satisfy their cholesterol requirement from receptormediated uptake ofplasma lipoproteins and from endogenous synthesis, while preventing sterol overaccumulation during periods of reduced demand (3).In recent studies a group of SRE-1-binding proteins (SREBPs) were isolated by DNA affinity chromatography from nuclear extracts of human HeLa cells (2, 4). Upon SDS/ polyacrylamide gel electrophoresis, the proteins clustered in the range 59-68 kDa. The specificity of DNA binding was confirmed by the demonstration that binding correlated with transcriptional activity in a series of 16 point mutants in the SRE-1 and surrounding sequences. Each of the proteins bound to the 7 point mutants that were transcribed in vivo, but they failed to bind to 9 point mutants that were not transcribed (2, 4).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Sequences of six peptides were obtained from a mixed preparation of SREBPs, and a cDNA that encoded a protei...