Abstract
The breakdown of stored fat deposits into its components is a highly regulated process that maintains plasma levels of free fatty acids to supply energy to cells. Insulin-mediated transcription of Atgl, the enzyme that mediates the rate-limiting step in lipolysis, is a key point of this regulation. Under conditions such as obesity or insulin resistance, Atgl transcription is often misregulated, which can contribute to overall disease progression. The mechanisms by which Atgl is induced during adipogenesis are not fully understood. We utilized computational approaches to identify putative transcriptional regulatory elements in Atgl and then tested the effect of these elements and the transcription factors that bind to them in cultured preadipocytes and mature adipocytes. Here we report that Atgl is down-regulated by the basal transcription factor Sp1 in preadipocytes and that the magnitude of down-regulation depends on interactions between Sp1 and peroxisome proliferator-activated receptor γ (PPARγ). In mature adipocytes, when PPARγ is abundant, PPARγ abrogated transcriptional repression by Sp1 at the Atgl promoter and up-regulated Atgl mRNA expression. Targeting the PPARγ-Sp1 interaction could be a potential therapeutic strategy to restore insulin sensitivity by modulating Atgl levels in adipocytes.
Highlights
The breakdown of stored fat deposits into its components is a highly regulated process that maintains plasma levels of free fatty acids to supply energy to cells
When 3T3-L1 preadipocytes were transfected with variable-length Atgl promoter-luciferase reporter constructs and incubated with mithramycin A, an Sp1 inhibitor, for 24 h, it abrogated the inhibitory effect of endogenous Sp1 when transfected with the Ϫ192/ϩ21 and Ϫ373/ϩ21 constructs contain
Our results showed that the shortest fragment of the Atgl promoter (Ϫ192/ϩ21) contains an evolutionarily conserved Sp1-binding site that is capable of inhibiting Atgl transcription when Sp1 is overexpressed exogenously
Summary
Analysis of approximately 3 kb of the DNA sequence immediately upstream of the Atgl transcription start site by Genomatix and TRANSFAC matrices revealed a putative Sp1-binding site at the minimal promoter (Ϫ50 to Ϫ36 bp). This effect is lost in the minimal promoter lacking the PPAR␥-binding site (Fig. 4B) and is in contrast to the data from preadipocytes, in which mutation of the Sp1 site in the fulllength promoter increased expression of the Atgl reporter construct (Fig. 3A) Together, this suggests that PPAR␥-mediated transactivation of Atgl is partially driven by the Sp1-binding site and that PPAR␥ binding to the promoter is essential to positively drive transcription of Atgl in mature adipocytes. This suggests that PPAR␥-mediated transactivation of Atgl is partially driven by the Sp1-binding site and that PPAR␥ binding to the promoter is essential to positively drive transcription of Atgl in mature adipocytes This regulation is cell-type specific, as a luciferase reporter assay performed using Sp1 overexpression vectors and the fulllength Atgl promoter construct in MEF and C2C12 cells did not result in the reduction in luciferase activity that was detected in preadipocytes These data support the hypothesis that Sp1 and PPAR␥ coordinately directly regulate Atgl transcription in a cell type- and stage-dependent manner that is governed by the changes in PPAR␥ abundance that occur during adipocyte differentiation (Fig. 5)
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