Abstract
The farnesoid X receptor (FXR, NR1H4) is a bile acid-responsive nuclear receptor that plays critical roles in the transcriptional regulation genes involved in cholesterol, bile acid, triglyceride, and carbohydrate metabolism. By microarray analysis of hepatic genes from female Zucker diabetic fatty (ZDF) rats treated with the FXR agonist GW4064, we have identified dimethylarginine dimethylaminohydrolase-1 (DDAH1) as an FXR target gene. DDAH1 is a key catabolic enzyme of asymmetric dimethylarginine (ADMA), a major endogenous nitric-oxide synthase inhibitor. Sequence analysis of the DDAH1 gene reveals the presence of an FXR response element (FXRE) located 90 kb downstream of the transcription initiation site and within the first intron. Functional analysis of the putative FXRE demonstrated GW4064 dose-dependent transcriptional activation from the element, and we have demonstrated that the FXRE sequence binds the FXR-RXR heterodimer. In vivo administration of GW4064 to female ZDF rats promoted a dose-dependent and >6-fold increase in hepatic DDAH1 gene expression. The level of serum ADMA was reduced concomitantly. These findings provide a mechanism by which FXR may increase endothelium-derived nitric oxide levels through modulation of serum ADMA levels via direct regulation of hepatic DDAH1 gene expression. Thus, beneficial clinical outcomes of FXR agonist therapy may include prevention of atherosclerosis and improvement of the metabolic syndrome.
Highlights
Nuclear receptors are transcription factors that serve as intracellular sensors for endocrine hormones and lipid metabolites, such as bile acids, fatty acids, oxysterols, and xenobiotics
We have shown that GW4064 increases hepatic expression of the dimethylarginine dimethylaminohydrolase-1 (DDAH1) gene that is accompanied by a reduction in serum asymmetric dimethylarginine (ADMA), a substrate of DDAH1 enzyme [28, 29]
In an attempt to study the hepatic pharmacology of FXR agonist GW4064, liver gene expression profiles from insulinresistant female Zucker diabetic fatty (ZDF) rats treated for 9 days with doses of GW4064 ranging from 10 –100 mg/kg were analyzed
Summary
Animal Care and Treatment—Female ZDF rats were purchased from Charles River (Wilmington, MA). Real-time quantitative PCR primer-probe sets for rat DDAH1 (Rn00574200_m1), rat SHP (Rn00589173_m1), rat PEPCK (Rn001529008_g1), rat glucose-6phosphatase (Rn00565347_m1) and rat 36B4 (Rn00821065_g1) were obtained from Taqman Assays-on-demand Gene Expression Products (PerkinElmer Life Sciences). Data are shown as mean Ϯ S.E. Reporter Constructs—A 470-bp DNA sequence (90 kb downstream from the transcription initiation site) containing the conserved FXRE in the DDAH1 intron 1 region was PCR-amplified from rat genomic DNA using primers 5Ј-CGCCGTGTACCCAGTTTTATGTCTA-3Ј and 5Ј-CTTAATACCTCATGGATCCCGACCT-3Ј. Study samples were derivatized with ortho-phthaldehyde as previously described [32], injected onto a 3.0 ϫ 150-mm Luna C18 [2] column equipped with a 4-mm precolumn (Phenomenex, Torrance, CA), and eluted under isocratic conditions (8.7% acetonitrile in 50 mM potassium phosphate, pH 6.5). In the case of the latter, this was referred to as the NOS impairment index
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