Evidence of Increased Glyceraldehyde-3-phosphate Dehydrogenase and Fatty Acid Synthetase Promoter Activities in Transiently Transfected Adipocytes from Genetically Obese Rats

Abstract

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.

Insulin and Sterol-regulatory Element-binding Protein-1c (SREBP-1C) Regulation of Gene Expression in 3T3-L1 Adipocytes

“…Differentiated 3T3-L1 cells were placed in serum-free medium containing 2% bovine serum albumin and no insulin for 24 h and transfected by electroporation as described previously for mature adipocytes (20). Briefly, 1-2 ϫ 10 6 cells in 200 l were shocked electrically in the presence of 20 g of promoter luciferase constructs and 1 g of Rous sarcoma virus-chloramphenicol acetyltransferase internal control.…”

Transcriptional Regulation of Adipocyte Hormone-Sensitive Lipase by Glucose

“…Primary rat white adipocytes were isolated by collagenase digestion of periepididymal fat pads from young (150 -200 g) male Wistar rats. Luciferase construct (20 g) and 5 g of pCMVlacZ (gift of P. Furth, University of Maryland, Baltimore, MD) were introduced into rat primary adipocytes by electroporation using a Biorad apparatus and cultured as previously described (20). ␤-Galactosidase expression was used to normalize transfection efficiency and was quantified using an o-nitrophenyl-␤-D-galactopyranoside colorimetric assay (18).…”

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

“…GAPDH concentrations vary significantly between different individuals , during pregnancy (Cale et al 1997), with developmental stage (Puissant et al 1994, Calvo et al 1997, during the cell cycle (Mansur et al 1993) and after the addition of the tumour promoter 12-Otetradecanoyl-phorbol-13-acetate (Spanakis 1993), dexamethasone (Oikarinen et al 1991) and carbon tetrachloride (Goldsworthy et al 1993). Insulin stimulates GAPDH transcription (Rolland et al 1995, Barroso et al 1999) through multiple insulinresponsive elements in its promoter (Alexander et al 1988, Nasrin et al 1990), at least one of which is recognised by C/EBP (Alexander-Bridges et al 1992). Similarly, the calcium ionophore A23187 induces GAPDH transcription through specific motifs in its promoter (Chao et al 1990).…”