Differential Feedback Regulation of Δ\u3csup\u3e4\u3c/sup\u3e-3- Oxosteroid 5β-Reductase Expression by Bile Acids

Δ4-3-oxosteroid 5β-reductase is member D1 of the aldo-keto reductase family 1 (AKR1D1), which catalyzes 5β-reduction of molecules with a 3-oxo-4-ene structure. Bile acid intermediates and most of the steroid hormones carry the 3-oxo-4-ene structure. Therefore, AKR1D1 plays critical roles in both bile acid synthesis and steroid hormone metabolism. Currently our understanding on transcriptional regulation of AKR1D1 under physiological and pathological conditions is very limited. In this study, we investigated the regulatory effects of primary bile acids, chenodeoxycholic acid (CDCA) and cholic acid (CA), on AKR1D1 expression. The expression levels of AKR1D1 mRNA and protein in vitro and in vivo following bile acid treatments were determined by real-time PCR and Western blotting. We found that CDCA markedly repressed AKR1D1 expression in vitro in human hepatoma HepG2 cells and in vivo in mice. On the contrary, CA significantly upregulated AKR1D1 expression in HepG2 cells and in mice. Further mechanistic investigations revealed that the farnesoid x receptor (FXR) signaling pathway was not involved in regulating AKR1D1 by bile acids. Instead, CDCA and CA regulated AKR1D1 through the mitogen-activated protein kinases/c-Jun N-terminal kinases (MAPK/JNK) signaling pathway. Inhibition of the MAPK/JNK pathway effectively abolished CDCA and CA-mediated regulation of AKR1D1. It was thus determined that AKR1D1 expression was regulated by CDCA and CA through modulating the MAPK/JNK signaling pathway. In conclusion, AKR1D1 expression was differentially regulated by primary bile acids through negative and positive feedback mechanisms. The findings indicated that both bile acid concentrations and compositions play important roles in regulating AKR1D1 expression, and consequently bile acid synthesis and steroid hormone metabolism

Inhibition of MAPK/JNK signaling pathway abolished CA-mediated regulation of AKR1D1.

<p>(A) HepG2 cells were treated with CA (50μM) in the absence or presence of MAPK/JNK inhibitor SP600125 (1μM), MAPK/ERK1/2 inhibitor PD98059 (5μM) or vehicle for 30 hrs, followed by detection of AKR1D1 mRNA by real-time PCR and (B) AKR1D1 protein by Western blot. (C) quantification of AKR1D1 protein levels in (B). One-way ANOVA was applied to analyze the data, followed by Tukey post-hoc test for multiple comparisons. * p<0.05.</p

CDCA and CA differentially modulated the MAPK/JNK signaling pathway.

<p>HepG2 cells were reversely transfected with 45 signaling pathway element reporter plasmids, followed by treatment of transfected cells with CDCA (25μM), CA (25μM) or vehicle DMSO (0.1%) for 30 h. The luciferase activities were detected with the Dual Luciferase Assays. The data are presented as mean ± SD of at least three separate experiments. One-way ANOVA was applied to analyze data with multiple groups, followed by Tukey post-hoc test for multiple comparisons. * p<0.05 and ** p<0.01.</p

FXR signaling was not involved in regulating AKR1D1 by bile acids in HepG2 cells.

<p>(A) HepG2 cells were treated with FXR agonist GW4064 (1μM) or vehicle DMSO (0.1%) for 30h, followed by detection of AKR1D1 and BSEP mRNA levels by real-time PCR. (B) HepG2 cells were transfected with FXRα1, FXRα2 or vector, followed by treatment with GW4064 (1μM) for 30h. The expression levels of AKR1D1 and BSEP were detected by real-time PCR. The Student’s t-test was applied to pair-wise comparison. One-way ANOVA was applied to analyze data with multiple groups, followed by Tukey post-hoc test for multiple comparisons. ** p<0.01.</p

Dysregulation of Δ \u3csup\u3e4\u3c/sup\u3e -3-oxosteroid 5β-reductase in diabetic patients: Implications and mechanisms

Aldo-keto reductase family 1 member D1 (AKR1D1) is a Δ 4 -3-oxosteroid 5β-reductase required for bile acid synthesis and steroid hormone metabolism. Both bile acids and steroid hormones, especially glucocorticoids, play important roles in regulating body metabolism and energy expenditure. Currently, our understanding on AKR1D1 regulation and its roles in metabolic diseases is limited. We found that AKR1D1 expression was markedly repressed in diabetic patients. Consistent with repressed AKR1D1 expression, hepatic bile acids were significantly reduced in diabetic patients. Mechanistic studies showed that activation of peroxisome proliferator-activated receptor-α (PPARα) transcriptionally down-regulated AKR1D1 expression in vitro in HepG2 cells and in vivo in mice. Consistently, PPARα signaling was enhanced in diabetic patients. In summary, dysregulation of AKR1D1 disrupted bile acid and steroid hormone homeostasis, which may contribute to the pathogenesis of diabetes. Restoring bile acid and steroid hormone homeostasis by modulating AKR1D1 expression may represent a new approach to develop therapies for diabetes

FXR signaling was not involved in regulating AKR1D1 by bile acids <i>in vivo</i> in mice.

<p>(A) two groups of mice (n = 6/group) were treated with GW4064 (5mg/kg) or vehicle propanediol through intraperitoneal injection twice a day for 3 days. The expression levels of Akr1d1 and Bsep were quantified by real-time PCR and (B) Western blot. (C) quantification of Akr1d1 and (D) Bsep protein levels in (B). (E) the expression levels of Akr1d1 and Bsep in wt and FXR-knockout (FXR-/-) mice were determined by real-time PCR and (F) Western blot. (G) quantification of Akr1d1 and (H) Bsep protein levels in (F). The data are presented as mean ± SD of the groups of mice. The Student’s t-test was applied to pair-wise comparison. ** p<0.01.</p

Inhibition of MAPK/JNK signaling pathway abolished CDCA-mediated regulation of AKR1D1.

<p>(A) HepG2 cells were treated with CDCA (25μM) in the absence or presence of MAPK/JNK inhibitor SP600125 (1μM), MAPK/ERK1/2 inhibitor PD98059 (5μM) or vehicle for 30 hrs, followed by detection of AKR1D1 mRNA by real-time PCR and (B) AKR1D1 protein by Western blot. (C) quantification of AKR1D1 protein levels in (B). One-way ANOVA was applied to analyze the data, followed by Tukey post-hoc test for multiple comparisons. * p<0.05 and ** p<0.01.</p

CA modulated AKR1D1 expression in time and dose-dependent manners in HepG2 cells.

<p>(A) HepG2 cells were treated with either CA (25 μM) or vehicle DMSO for 8, 24 or 30 hrs, followed by detection of AKR1D1 mRNA with real-time PCR. (B) HepG2 cells were treated with various concentrations of CA (0, 5, 25, 50, 100, 200 μM) for 30 hrs, followed by detection of AKR1D1 mRNA expression with real-time PCR. *p<0.05 with the Student’s t-test for pair-wise comparison.</p