The ability to regulate specific genes of energy metabolism in response to fasting and feeding is an important adaptation allowing survival of intermittent food supplies. However, little is known about transcription factors involved in such responses in higher organisms. We show here that gene expression in adipose tissue for adipocyte determination differentiation dependent factor (ADD) 1/sterol regulatory element binding protein (SREBP) 1, a basic-helix-loop-helix protein that has a dual DNA-binding specificity, is reduced dramatically upon fasting and elevated upon refeeding; this parallels closely the regulation of two adipose cell genes that are crucial in energy homeostasis, fatty acid synthetase (FAS) and leptin. This elevation of ADD1/SREBP1, leptin, and FAS that is induced by feeding in vivo is mimicked by exposure of cultured adipocytes to insulin, the classic hormone of the fed state. We also show that the promoters for both leptin and FAS are transactivated by ADD1/SREBP1. A mutation in the basic domain of ADD1/SREBP1 that allows E-box binding but destroys sterol regulatory element-1 binding prevents leptin gene transactivation but has no effect on the increase in FAS promoter function. Molecular dissection of the FAS promoter shows that most if not all of this action of ADD1/SREBP1 is through an E-box motif at -64 to -59, contained with a sequence identified previously as the major insulin response element of this gene. These results indicate that ADD1/SREBP1 is a key transcription factor linking changes in nutritional status and insulin levels to the expression of certain genes that regulate systemic energy metabolism.

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The UCP2-UCP3 gene cluster maps to chromosome 11q13 in humans, and polymorphisms in these genes may contribute to obesity through effects on energy metabolism. DNA sequencing of UCP2 and UCP3 revealed three polymorphisms informative for association studies: an Ala-->Val substitution in exon 4 of UCP2, a 45 bp insertion/deletion in the 3'-untranslated region of exon 8 of UCP2 and a C-->T silent polymorphism in exon 3 of UCP3. Initially, 82 young (mean age = 30 +/- 7 years), unrelated, full-blooded, non-diabetic Pima Indians were typed for these polymorphisms by direct sequencing. The three sites were in linkage disequilibrium ( P < 0.00001). The UCP2 variants were associated with metabolic rate during sleep (exon 4, P = 0.007; exon 8, P = 0.016) and over 24 h (exon 8, P = 0.038). Heterozygotes for UCP2 variants had higher metabolic rates than homozygotes. The UCP3 variant was not significantly associated with metabolic rate or obesity. In a further 790 full-blooded Pima Indians, there was no significant association between the insertion/deletion polymorphism and body mass index (BMI). However, when only individuals >45 years of age were considered, heterozygotes (subjects with the highest sleeping metabolic rate) had the lowest BMI (P = 0.04). The location of the insertion/deletion polymorphism suggested a role in mRNA stability; however, it appeared to have no effect on skeletal muscle UCP2 mRNA levels in a subset of 23 randomly chosen Pima Indians. In conclusion, these results suggest a contribution from UCP2 (or UCP3) to variation in metabolic rate in young Pima Indians which may contribute to overall body fat content later in life.

Peroxisome proliferator-activated receptor ␥ (PPAR␥) is a member of the nuclear hormone receptor superfamily, and is an important regulator of adipogenesis and adipocyte gene expression. PPAR␥ exists as two isoforms, PPAR␥1 and PPAR␥2, that differ only in their N termini. Both isoforms are activated by ligands that include the antidiabetic thiazoladinedione drugs and 15-deoxy-⌬ 12, 14 -prostaglandin J2, and potential differences in their function have yet to be described. We report that, in addition to a ligand-activated transcriptional activity, when studied under conditions of ligand depletion, intact PPAR␥ has a ligand-independent activation domain. To identify the basis for this ligand-independent activation, we used GAL4-PPAR␥ chimeric expression constructs and UAS-TK-LUC in CV1 cells and isolated rat adipocytes. In both cell systems, isolated PPAR␥1 and PPAR␥2 N termini have activation domains, and the activation function of PPAR␥2 is 5-6-fold greater than that of PPAR␥1. Insulin enhances the transcriptional effect mediated by both PPAR␥1 and PPAR␥2 N-terminal domains. These data demonstrate that 1) PPAR␥ has an N-terminal (ligandindependent) activation domain; 2) PPAR␥1 and PPAR␥2 N termini have distinct activation capacities; and 3) insulin can potentiate the activity of the N-terminal domain of PPAR␥.The peroxisome proliferator-activated receptor ␥ (PPAR␥) 1 is a member of the nuclear receptor superfamily that plays a pivotal role in the molecular determination of adipogenesis and the regulation of adipocyte gene expression (1-5). Under appropriate conditions, expression of PPAR␥ through retroviral infection of fibroblastic cell lines is sufficient to cause differentiation along an adipocyte lineage, as assessed by expression of adipocyte-specific genes, accumulation of lipid, and acquisition of adipocyte morphology (6). Recently, it has been shown that 15-deoxy-⌬ 12, 14 -prostaglandin J2 (PG J2) is a high affinity ligand for PPAR␥ (7,8) and that PPAR␥ is also the receptor for the thiazoladinedione class of insulin-sensitizing drugs (7,8). PPAR␥ resembles other members of the nuclear receptor superfamily in that ligand-dependent receptor activation alters the rates of transcription of genes, specifically those that have peroxisome proliferator response elements (PPREs) within their promoters (e.g. aP2, phosphoenolpyruvate carboxykinase, and uncoupling protein) (9 -11).PPAR␥ exists as two isoforms, PPAR␥1 and PPAR␥2, that differ only in their N termini, with PPAR␥2 having an additional 30 amino acids that are encoded by a single exon (9, 12). Expression of mRNA encoding the two isoforms is driven by alternative promoters within a single PPAR␥ gene (12), and their expression is differentially regulated in a tissue-specific manner. PPAR␥2 is most abundantly expressed in adipocytes and is relatively specific for this tissue (9, 13). In contrast, while PPAR␥1 is also expressed at a high level in adipocytes, it is also found at significant but lower levels in a number of other tissues, including muscle (13-15...