The transcriptional regulation of the human angiotensinogen gene after high-fat diet is haplotype-dependent: Novel insights into the gene-regulatory networks and implications for human hypertension

Figures

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  Fig 1. TG-mice with Hap I have increased expression of the hAGT gene after HFD, as compared with Hap II. Human AGT expression is significantly elevated after HFD in TG mice with Hap I than Hap II, in adipose and liver tissues. Change in mRNA expression of the hAGT gene after 12 weeks of HFD, as compared to baseline CD, in adipose (A) and liver tissue (B). mAGT expression in adipose (C) and liver tissue (D) of TG mice fed with control diet (CD) or HFD in both haplotypes. mRNA was determined by quantitative RT-PCR analysis. Results are shown as mean ±SEM (error bars) from n = 4 per group. *p ≤ 0.05 versus Hap II with HFD; # p ≤0.05 versus CD in both Hap I & Hap II.

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  Fig 2. Transcription factor binding sites in the hAGT gene promoter. Nucleotide sequence of different regions of the promoter of the hAGT gene along with position of SNPs (marked by asterisks). Variants in Hap II are shown in red, and in Hap I are shown in black. Consensus binding sites of different transcription factors are shown below the nucleotide sequence of the promoter.

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  Fig 3. Expression of the transcription factors associated with the regulation of the hAGT, with or without HFD. Expression of transcription factors, GR, CEBPβ, and STAT3 with or without HFD in adipose (A) and liver (B) tissue. Expression of mRNA was calculated for the HFD and CD group and normalized by the respective GAPDH values. Results are shown as mean ± S.E. (error bars) (n = 4). *p ≤0.05 versus Hap I & Hap II with CD.

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  Fig 4. ChIP assay on the -217 and -1329 regions of the hAGT gene from the chromatin obtained from adipose tissue of TG mice after HFD. ChIP assay was performed by PCR amplification of the immunoprecipitated DNA in the presence of antibodies against GR (a), CEBPβ (b) and STAT3 (c), input DNA (d), IgG (e), and nonspecific primers (NS) (f). Immunoprecipitated DNA was used to amplify nucleotide sequence encompassing either -217 region (Fig 4A) or -1329 region (Fig 4B) of the hAGT gene as described in “Materials and Methods.” Quantitation of GR, CEBPβ and STAT3 -enriched DNA, relative to input, at the -217 region or -1329 region of the hAGT gene was performed by Q-PCR. Result shows a significant increase in the HFD-induced GR, CEBPβ and STAT3 binding in TG mice with Hap I as compared to Hap II. *, p≤0.05 versus Hap II with HFD. Results are shown as mean ± S.E. (n = 4). A.U., arbitrary units.

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  Fig 5. Effect of HFD on plasma level of hAGT in Hap I and Hap II TG mice. Plasma levels of hAGT in TG mice containing either Hap I or Hap II of the hAGT gene in CD or HFD fed TG mice. Results are shown as mean ± SEM from n = 4. *p≤0.05 versus Hap II with HFD; # p≤0.05 versus Hap II with CD.