Expression of the peroxisome proliferator activated receptor gamma gene is repressed by DNA methylation in visceral adipose tissue of mouse models of diabetes

Abstract

Background: Adipose tissues serve not only as a store for energy in the form of lipid, but also as endocrine tissues that regulates metabolic activities of the organism by secreting various kinds of hormones. Peroxisome proliferator activated receptor gamma (PPARgamma) is a key regulator of adipocyte differentiation that induces the expression of adipocyte-specific genes in preadipocytes and mediates their differentiation into adipocytes. Furthermore, PPARgamma has an important role to maintain the physiological function of mature adipocyte by controlling expressions of various genes properly. Therefore, any reduction in amount and activity of PPARgamma is linked to the pathogenesis of metabolic syndrome.

Results: In this study, we investigated the contribution of epigenetic transcriptional regulatory mechanisms, such as DNA methylation, to the expression of the PPARgamma gene, and further evaluated the contribution of such epigenetic regulatory mechanisms to the pathogenesis of metabolic syndrome. In 3T3-L1 preadipocytes, the promoter of the PPARgamma2 gene was hypermethylated, but was progressively demethylated upon induction of differentiation, which was accompanied by an increase of mRNA expression. Moreover, treatment of cells with 5'-aza-cytideine, an inhibitor of DNA methylation, increased expression of the PPARgamma gene in a dose-dependent manner. Methylation in vitro of a PPARgamma promoter-driven reporter construct also repressed the transcription of a downstream reporter gene. These results suggest that the expression of the PPARgamma gene is inhibited by methylation of its promoter. We next compared the methylation status of the PPARgamma promoters in adipocytes from wild-type (WT) mice with those from two diabetic mouse models: +Leprdb/+Leprdb and diet-induced obesity mice. Interestingly, we found increased methylation of the PPARgamma promoter in visceral adipose tissues (VAT) of the mouse models of diabetes, compared to that observed in wild-type mice. We observed a concomitant decrease in the level of PPARgamma mRNA in the diabetic mice compared to the WT mice.

Conclusion: We conclude that the expression of PPARgamma gene is regulated by DNA methylation of its promoter region and propose that reduced expression of PPARgamma owing to DNA methylation in adipocytes of the VAT may contribute to the pathogenesis of metabolic syndrome.

Figures

Figure 1

Expression of peroxisome proliferators activated receptor γ (PPARγ) mRNA and differential methylation of the PPARγ promoter. (a) Relative expression of PPARγ mRNA in NIH/3T3, 3T3-L1 preadipocytes (day 0) and differentiated 3T3-L1 adipocytes (day 6). The expression of PPARγ and β-actin mRNAs were determined by real time reverse transcriptase polymerase chain reaction (RT-PCR). The level of PPARγ mRNA was normalized to that of β-actin, and the relative normalized levels are shown (n = 3, mean ± SD). (b) A schematic diagram of the PPARγ promoter. An arrow indicates the transcription start site (TSS) (+1 bp), and short vertical lines indicate the positions of the methylation sites relative to the TSS. The region detected by the chromatin immunoprecipitation (ChIP) analysis is indicated below the schematic (see Figure 4). (c) Bisulfite sequencing analysis of the DNA methylation profile of the individual CpG sites in the PPARγ promoter in NIH/3T3, 3T3-L1 preadipocytes (day 0) and 3T3-L1 adipocytes (day 6). Each PCR product was subcloned, and eight clones were subjected to sequencing analysis. The data represent the aggregate total of three independent experiments. The methylation status of each site, either methylated (closed circle) or unmethylated (open circle), is aligned corresponding to their genomic order (represented at the bottom of the results for NIH/3T3 cells).

Figure 2

Activation of peroxisome proliferators activated receptor γ (PPARγ) gene by 5'-aza-cytideine (5'-aza-C) and evaluation of promoter activity with a luciferase assay. (a, b) Expression of PPARγ mRNA in 5'-aza-C-treated NIH/3T3 and 3T3-L1 preadipocytes. The cells were cultured in growth medium containing 5'-aza-C at the indicated concentrations for 48 h before harvest. The mRNA expression levels were determined by real time reverse transcriptase polymerase chain reaction (RT-PCR) and normalized to the levels of β-actin mRNA measured in parallel experiments (n = 3, mean ± SD). The expression level of PPARγ mRNA in differentiated day 6 3T3-L1 adipocytes was also presented at the right of (b) for comparison. (c) Luciferase expression from PPARγ promoter reporter constructs in the presence or absence of in vitro DNA methylation in NIH/3T3, 3T3-L1 preadipocytes and differentiating adipocytes (day 4). Approximately 1 kb of the region upstream of the PPARγ transcription start site (TSS) was cloned into a luciferase reporter vector, and the vector was methylated in vitro as needed. Relative luciferase activity, normalized to the activity of a cotransfected internal control vector, is shown (n = 3, mean ± SD).

Figure 3

Kinetic analysis of peroxisome proliferators activated receptor γ (PPARγ) promoter demethylation, mRNA expression and cell proliferation. (a, b) The timelapse analysis of PPARγ promoter demethylation and mRNA expression during adipogenesis. Preadipocytes were stimulated to differentiate on day 0 (D0), and harvested every 24 or 48 h until day 8 (D8). The methylation status of the -437 bp, -298 bp and -247 bp CpG sites were determined by restriction endonuclease digestion, and the fraction of the promoter fragments in which all of the three sites were unmethylated is represented (a). The mRNA expression levels were measured by real time reverse transcriptase polymerase chain reaction (RT-PCR) and normalized to the level of β-actin mRNA (b). Individual assessments were repeated three times and the means ± SD are represented, respectively. (c) The increase in cell number by mitotic clonal expansion. The number of differentiating cells was counted at the indicated timepoints, and the cell density was calculated.

Figure 4

Chromatin immunoprecipitation (ChIP) assays of the peroxisome proliferators activated receptor γ (PPARγ) promoter region during adipogenesis. 3T3-L1 cells were harvested at the indicated times, and 106 cells were used as the input for each assay. DNA fragments immunoprecipitated by the indicated antibody were recovered and amplified by the primers designed for the PPARγ promoter region (see Figure 1). A total of 1% of the input was also amplified without ChIP, and is shown at the bottom.

Figure 5

Comparison of the DNA methylation profile of the peroxisome proliferators activated receptor γ (PPARγ) in white adipose tissue (WAT). Genomic DNA was extracted from subcutaneous adipose tissue (SAT) (a, c) and epididymal adipose tissues (EAT) (b, d) of 10 week-old wild-type (WT) or db/db mice (a, b) or 20 week-old WT/diet-induced obesity mice (c, d). Genomic DNA prepared from each tissue was treated with sodium bisulfite, and amplified by polymerase chain reaction (PCR) with the primers designed for the flanking regions of the -437 bp or -247 bp CpG site. The methylation status of each site was estimated by the efficiency of restriction endonuclease digestion of the PCR amplicon, and the percentages of the methylated fragments are represented (n = 3, mean ± SD, *P < 0.05, t test).

Figure 6

Expression levels of peroxisome proliferators activated receptor γ (PPARγ) mRNA in white adipose tissues of normal and diabetic mice. Total RNA was extracted from subcutaneous adipose tissue (SAT) (a) and epididymal adipose tissues (EAT) (b) of 10 week-old wild-type (WT) or db/db mice, and 20 week-old WT or diet-induced obesity mice. The mRNA expression levels of PPARγ2 were determined by real time reverse transcriptase polymerase chain reaction (RT-PCR), and normalized to that of β-actin measured in parallel as internal controls. Data represent the relative values of the mean ± SD of three independent experiments performed in triplicate.