Previous studies have shown that the adipose tissue of young genetically obese Zucker rats was characterized by a coordinate overtranscription of lipogenic genes, suggesting that the fa mutation triggers transcription factor(s) acting in common on the promoters of these genes. To test this hypothesis, we developed a system of transient transfection of rat adipocytes with constructs containing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fatty acid synthetase (FAS) promoters fused to gene reporter CAT. Those transfected cells expressed high levels of promoter-driven chloramphenicol acetyltransferase (CAT) activity through correctly initiated transcription as shown by primer extension analysis. Using this system we found a direct effect of insulin on GAPDH and FAS gene expression in rat adipocytes. In transfected adipocytes from obese compared to lean rats, activity of GAPDH and FAS promoters fused to CAT, was 2.6- and 8-fold increased, respectively. In contrast when reporter gene activity was driven by either phosphoenolpyruvate carboxykinase or beta-actin promoter, no difference could be observed between lean and obese, pointing out the promoter specificity of genotype effect. 5' deletion analysis of GAPDH promoter allowed us to narrow down the fa responsive region to nucleotide -488-329. As assessed by gel retardation and DNase I footprinting analysis, adipocyte nuclear protein interactions to this 159-bp fragment were found to be identical and to footprint the same 20-bp sequence. This study pointed out that overexpression of GAPDH and FAS genes in adipose tissue of genetically obese rats relies on promoter activation, through a 159-bp cis-acting region within the GAPDH promoter. The effects of the fa mutation on trans-acting factors binding to this region remain to be identified.

We have previously shown that the proximal 2-kb sequence of the fatty acid synthase (FAS) promoter transfected into rat adipocytes was highly sensitive to the cellular context, displaying an overactivity in obese (fa/fa) versus lean Zucker rat adipocytes. Using deletional analysis, we show here that FAS promoter activity mainly depends on a region from -200 to -126. This sequence exerts a strong negative effect on FAS promoter in adipocytes from lean rats but not in those from obese rats, resulting in a marked overtranscriptional activity in the latter cells. This region, fused to a heterologous promoter, the E1b TATA box, induced differential levels of gene reporter activity in lean and obese rat adipocytes, indicating it harbors fa-responsive element(s). Whatever the rat genotype, adipocyte nuclear proteins were shown to footprint the same protected sequence within the fa-responsive region, and supershift analysis demonstrated that Sp1 or Sp1-like proteins were bound to this DNA subregion. Compelling evidence that the Sp1 binding site contained in this sequence was implicated in the differential promoter activity in lean versus obese rats, was provided by the observation that mutations at this Sp1 site induced a 2.5-fold increase in FAS promoter activity in adipocytes from lean rats, whereas they had no effect in adipocytes from obese rats.

ROLLAND, VIOLAINE, KARINE CLEMENT, ISA-BELLE DUGAIL, BERNARD GUY-GRAND, AR-NAUD BASDEVANT, PHILIPPE FROGUEL, MAR-CELLE LAVAU. Leptin receptor gene in a large cohort of massively obese subjects: No indication of the fdfa rat mutation. Detection of an intronic variant with no association with obesity. Obes Res. 1998;6:122-127. The massive obesity caused in rodents by the disruption of the leptin-receptor signal through genetic defects at the level of either leptin (OB) or leptin receptor (OB-R) has raised the question of the relevance of these genes to morbid obesity in humans. In this study, we screened a large population of massively obese subjects for the presence of a leptin receptor mutation homologous to that of fdfa rats, a single base substitution changing glutamine 269, a highly conserved glutamine found at position 270 in the human sequence. After polymerase chain reaction (PCR) amplification of a DNA region encompassing the end of exon 5, intron 5, and the beginning of exon 6, we performed restriction fragment length polymorphism analysis. Within the limitations of this approach where only mutations introducing restriction sites (5 of 8 possibilities) could be assessed, no evidence of mutation at the codon gln 270 was found in 343 massively obese subjects. However, a new OB-R gene variant in intron 5 was revealed by Mae11 digestion of the PCR products. MueIYhOB-R genotyping revealed no difference in the distribution of the genotypes between obese subjects and a group of 79 unrelated nonobese control subjects. In addition, no significant association between various obesity-related metabolic phenotypes and the presence of MueIYhOB-R alleles was found. Thus, our results did not support a significant role for the MaeIIhOB-R gene variant in the development of the obese phenotype in the population we studied.