Peroxisome proliferator-activated receptor-gamma coactivator-1alpha activation of CYP7A1 during food restriction and diabetes is still inhibited by small heterodimer partner

Cholesterol 7alpha-hydroxylase (CYP7A1) catalyzes the rate-limiting step in the classic pathway of hepatic bile acid biosynthesis from cholesterol. During fasting and in type I diabetes, elevated levels of peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1alpha) induce expression of the Cyp7A1 gene and overexpression of PGC-1alpha in hepatoma cells stimulates bile acid synthesis. Using Ad-PGC-1alpha-RNA interference to induce acute disruption of PGC-1alpha in mice, here we show that PGC-1alpha is necessary for fasting-mediated induction of CYP7A1. Co-immunoprecipitation and promoter activation studies reveal that the induction of CYP7A1 is mediated by direct interaction between PGC-1alpha and the AF2 domain of liver receptor homolog-1 (LRH-1). In contrast, the very similar PGC-1beta could not substitute for PGC-1alpha. We also show that transactivation of PGC-1alpha and LRH-1 is repressed by the small heterodimer partner (SHP). Treatment of mice with GW4064, a synthetic agonist for farnesoid X receptor, induced SHP expression and decreased both the recruitment of PGC-1alpha to the Cyp7A1 promoter and the fasting-induced expression of CYP7A1 mRNA. These data suggest that PGC-1alpha is an important co-activator for LRH-1 and that SHP targets the interaction between LRH-1 and PGC-1alpha to inhibit CYP7A1 expression. Overall, these studies provide further evidence for the important role of PGC-1alpha in bile acid homeostasis and suggest that pharmacological targeting of farnesoid X receptor in vivo can be used to reverse the increase in CYP7A1 associated with adverse metabolic conditions.     

FXR-mediated down-regulation of CYP7A1 dominates LXRalpha in long-term cholesterol-fed NZW rabbits

We investigated how cholesterol feeding regulates cholesterol 7alpha-hydroxylase (CYP7A1) via the nuclear receptors farnesoid X receptor (FXR) and liver X receptor alpha (LXRalpha) in New Zealand white rabbits. After 1 day of 2% cholesterol feeding, when the bile acid pool size had not expanded, mRNA levels of the FXR target genes short-heterodimer partner (SHP) and sterol 12alpha-hydroxylase (CYP8B) were unchanged, indicating that FXR activation remained constant. In contrast, the mRNA levels of the LXRalpha target genes ATP binding cassette transporter A1 (ABCA1) and cholesteryl ester transfer protein (CETP) increased 5-fold and 2.3-fold, respectively, associated with significant increases in hepatic concentrations of oxysterols. Activity and mRNA levels of CYP7A1 increased 2.4 times and 2.2 times, respectively. After 10 days of cholesterol feeding, the bile acid pool size increased nearly 2-fold. SHP mRNA levels increased 4.1-fold while CYP8B declined 64%. ABCA1 mRNA rose 8-fold and CETP mRNA remained elevated. Activity and mRNA of CYP7A1 decreased 60% and 90%, respectively. Feeding cholesterol for 1 day did not enlarge the ligand pool size or change FXR activation, while LXRalpha was activated highly secondary to increased hepatic oxysterols. As a result, CYP7A1 was up-regulated. After 10 days of cholesterol feeding, the bile acid (FXR ligand) pool size increased, which activated FXR and inhibited CYP7A1 despite continued activation of LXRalpha. Thus, in rabbits, when FXR and LXRalpha are activated simultaneously, the inhibitory effect of FXR overrides the stimulatory effect of LXRalpha to suppress CYP7A1 mRNA expression.     

The small heterodimer partner interacts with the pregnane X receptor and represses its transcriptional activity

SHP (small heterodimer partner, NR1I0) is an atypical orphan member of the nuclear receptor subfamily in that it lacks a DNA-binding domain. It is mostly expressed in the liver, where it binds to and inhibits the function of nuclear receptors. SHP is up-regulated by primary bile acids, through the activation of their receptor farnesoid X receptor, leading to the repression of cholesterol 7alpha-hydroxylase (CYP7alpha) expression, the rate-limiting enzyme in bile acid production from cholesterol. PXR (pregnane X receptor, NR1I2) is a broad-specificity sensor that recognizes a wide variety of synthetic drugs as well as endogenous compounds such as bile acid precursors. Upon activation, PXR induces CYP3A and inhibits CYP7alpha, suggesting that PXR can act on both bile acid synthesis and elimination. Indeed, CYP7alpha and CYP3A are involved in biochemical pathways leading to cholesterol conversion into primary bile acids, whereas CYP3A is also involved in the detoxification of toxic secondary bile acid derivatives. Here, we show that PXR is a target for SHP. Using pull-down assays, we show that SHP interacts with both murine and human PXR in a ligand-dependent manner. From transient transfection assays, SHP is shown to be a potent repressor of PXR transactivation. Furthermore, we report that chenodeoxycholic acid and cholic acid, two farnesoid X receptor ligands, induce up-regulation of SHP and provoke a repression of PXR-mediated CYP3A induction in human hepatocytes as well as in vivo in mice. These results reveal an elaborate regulatory cascade, tightly controlled by SHP, for both the maintenance of bile acid production and detoxification in the liver.     

Change in hepatic and plasma bile acid contents and its regulatory gene expression in the chicken embryo

To investigate changes in bile acid biosynthesis in chicken (Gallus gallus) during embryonic stages, we studied the contribution of hepatic and plasma total bile acid levels, mRNA expression of cholesterol 7 alpha-hydroxylase (CYP7A1), and the expression of its regulatory genes in two embryo models (i.e., broilers and layers) differing in lipid metabolism. Total bile acid levels in plasma and liver were low during embryonic stages, as well as expression of CYP7A1. At hatch (P0), hepatic and plasma total bile acid levels and CYP7A1 mRNA expression in liver were markedly increased in both models. The hepatic mRNA expression of liver X receptor (LXR)alpha, a regulator of CYP7A1 gene expression gradually decreased with developmental stages of both broilers and layers. The hepatic mRNA expression of farnesoid X receptor (FXR), a repressor of CYP7A1 gene expression, also decreased with embryonic development. The present results showed that the mRNA expression of CYP7A1 and synthesis of bile acids was low in embryonic stages, suggesting that FXR might be a key regulator of CYP7A1 gene expression in the chicken embryo.     

Roles of nuclear receptors in the up-regulation of hepatic cholesterol 7alpha-hydroxylase by cholestyramine in rats

Nuclear receptors are involved in regulating the expression of cholesterol 7alpha-hydroxylase (CYP7A1), however, their roles in the up-regulation of CYP7A1 by cholestyramine (CSR) are still unclear. In the present study, male Wistar rats were divided into four groups and fed [high sucrose + 10% lard diet] (H), [H + 3% CSR diet] (H + CSR), [H + 0.5% cholesterol + 0.25% sodium cholate diet] (C), or [C + 3% CSR diet] (C + CSR) for 2 weeks. Cholestyramine decreased serum and liver cholesterol levels significantly in rats fed C-based diets, but had no effect on these parameters in rats fed H-based diets. Cholestyramine raised hepatic levels of CYP7A1 mRNA and activity in both groups. The gene expression of hepatic ATP-binding cassettes A1 and G5, regulated by liver X receptor (LXR), were unchanged and down-regulated by cholestyramine, respectively. The mRNA levels of the hepatic ATP-binding cassette B11 and short heterodimer partner (SHP), regulated by farnesoid X receptor (FXR), were not changed by cholestyramine. C-based diets, which contained cholesterol and cholic acid, increased SHP mRNA levels compared to H-based diets. Consequently, in rats fed the C+CSR diet, hepatic FXR was activated by dietary bile acids, but the hepatic CYP7A1 mRNA level was increased 16-fold compared to that in rats fed an H diet. These results suggest that cholestyramine up-regulates the expression of CYP7A1 independently via LXR- or FXR-mediated pathways in rats.     

Dietary cholesterol stimulates CYP7A1 in rats because farnesoid X receptor is not activated

Cholesterol feeding upregulates CYP7A1 in rats but downregulates CYP7A1 in rabbits. To clarify the mechanism responsible for the upregulation of CYP7A1 in cholesterol-fed rats, the effects of dietary cholesterol (Ch) and cholic acid (CA) on the activation of the nuclear receptors, liver X-receptor (LXR-alpha) and farsenoid X-receptor (FXR), which positively and negatively regulate CYP7A1, were investigated in rats. Studies were carried out in four groups (n = 12/group) of male Sprague-Dawley rats fed regular chow (control), 2% Ch, 2% Ch + 1% CA, and 1% CA alone for 1 wk. Changes in mRNA expression of short heterodimer partner (SHP) and bile salt export pump (BSEP), target genes for FXR, were determined to indicate FXR activation, whereas the expression of ABCA1 and lipoprotein lipase (LPL), target genes for LXR-alpha, reflected activation. CYP7A1 mRNA and activity increased twofold and 70%, respectively, in rats fed Ch alone when the bile acid pool size was stable but decreased 43 and 49%, respectively, after CA was added to the Ch diet, which expanded the bile acid pool 3.4-fold. SHP and BSEP mRNA levels did not change after feeding Ch but increased 88 and 37% in rats fed Ch + CA. This indicated that FXR was activated by the expanded bile acid pool. When Ch or Ch + CA were fed, hepatic concentrations of oxysterols, ligands for LXR-alpha increased to activate LXR-alpha, as evidenced by increased mRNA levels of ABCA1 and LPL. Feeding CA alone enlarged the bile acid pool threefold and increased the expression of both SHP and BSEP. These results suggest that LXR-alpha was activated in rats fed both Ch or Ch + CA, whereas CYP7A1 mRNA and activity were induced only in Ch-fed rats where the bile acid pool was not enlarged such that FXR was not activated. In rats fed Ch + CA, the bile acid pool expanded, which activated FXR to offset the stimulatory effects of LXR-alpha on CYP7A1.     

Bile acids reduce SR-BI expression in hepatocytes by a pathway involving FXR/RXR, SHP, and LRH-1

Hepatic SR-BI mediates uptake of circulating cholesterol into liver hepatocytes where a part of the cholesterol is metabolised to bile acids. In the hepatocytes, bile acids reduce their own synthesis by a negative feedback loop to prevent toxic high levels of bile acids. Bile acid-activated FXR/RXR represses expression of CYP7A1, the rate-limiting enzyme during bile acid synthesis, by inducing the expression of SHP, which inhibits LXR/RXR and LRH-1-transactivation of CYP7A1. The present paper presents data indicating that CDCA suppresses SR-BI expression by the same pathway. As previously reported, LRH-1 induces SR-BI promoter activity. Here we show that CDCA or over-expression of SHP inhibit this transactivation. No FXR-response element was identified in the bile acid-responsive region of the SR-BI promoter (-1200bp/-937bp). However, a binding site for LRH-1 was characterised and shown to specifically bind LRH-1. The present study shows that also the SR-BI-mediated supply of cholesterol, the substrate for bile acid synthesis, is feedback regulated by bile acids.