Liver X receptors (LXRs), LXR
and LXR
, are nuclear receptors that regulate the metabolism of cholesterol and bile acids and are activated by oxysterols. Humanized
(
) mice express the 9-human
genes ...associated with the
locus in a
-null background. The expression of UGT1A1 is developmentally delayed in the liver and intestines, resulting in the accumulation of serum bilirubin during the neonatal period. Induction of UGT1A1 in newborn
mice leads to rapid reduction in total serum bilirubin (TSB) levels, a phenotype measurement that allows for an accurate prediction on UGT1A1 expression. When neonatal
mice were treated by oral gavage with the LXR agonist T0901317, TSB levels were dramatically reduced. To determine the LXR contribution to the induction of UGT1A1 and the lowering of TSB levels, experiments were conducted in neonatal
,
, and
mice treated with T0901317. Induction of liver UGT1A1 was dependent upon LXR
, with the induction pattern paralleling induction of LXR
-specific stearoyl CoA desaturase 1. However, the actions of T0901317 were also shown to display a lack of specificity for LXR, with the induction of liver UGT1A1 in
mice, a result associated with activation of both pregnane X receptor and constitutive androstane receptor. However, the LXR agonist GW3965 was highly selective toward LXR
, showing no impact on lowering TSB values or inducing UGT1A1 in
mice. An LXR-specific enhancer site on the
gene was identified, along with convincing evidence that LXR
is crucial in maintaining constitutive expression of UGT1A1 in adult
mice. SIGNIFICANCE STATEMENT: It has been established that activation of LXR
, and not LXR
, is responsible for the induction of liver UGT1A1 and metabolism of serum bilirubin in neonatal
mice. Although induction of the human
gene is initiated at a newly characterized LXR enhancer site, allelic deletion of the
gene drastically reduces the constitutive expression of liver UGT1A1 in adult
mice. Combined, these findings indicate that LXR
is critical for the developmental expression of UGT1A1.
Cholesterol-derived bile acids (BAs) affect numerous physiological functions such as glucose homeostasis, lipid metabolism and absorption, intestinal inflammation and immunity, as well as intestinal ...microbiota diversity. Diet influences the composition of the BA pool. In the present study, we analyzed the impact of a dietary supplementation with a freeze-dried blueberry powder (BBP) on the fecal BA pool composition. The diet of 11 men and 13 women at risk of metabolic syndrome was supplemented with 50 g/day of BBP for 8 weeks, and feces were harvested before (pre) and after (post) BBP consumption. BAs were profiled using liquid chromatography coupled with tandem mass spectrometry. No significant changes in total BAs were detected when comparing pre- vs. post-BBP consumption samples. However, post-BBP consumption samples exhibited significant accumulations of glycine-conjugated BAs (
= 0.04), glycochenodeoxycholic (
= 0.01), and glycoursodeoxycholic (
= 0.01) acids, as well as a significant reduction (
= 0.03) in the secondary BA levels compared with pre-BBP feces. In conclusion, the fecal bileacidome is significantly altered after the consumption of BBP for 8 weeks. While additional studies are needed to fully understand the underlying mechanisms and physiological implications of these changes, our data suggest that the consumption of blueberries can modulate toxic BA elimination.
Ursodeoxycholic acid (UDCA) is the first line therapy for the treatment of cholestatic and autoimmune liver diseases. Its clinical use is currently limited by a significant proportion of ...non-responder patients. Polyunsaturated fatty acids (n-3 PUFAs) possess important anti-inflammatory properties and protect liver cells against bile acid (BA)-induced toxicity. The present study was designed to rapidly evaluate whether combining n-3 PUFAs (i.e., eicosapentaenoic EPA and docosahexaenoic DHA acids) to UDCA would provide additional benefits when compared to the drug alone. The parameters evaluated were (i) the expression of genes governing BA synthesis, transport, and metabolism; (ii) the prevention of BA-induced apoptosis and endoplasmic reticulum (ER)-stress; and (iii) the control of BA- and LPS-dependent inflammation. In the absence of n-3 PUFAs, most of the parameters investigated were unaffected by UDCA or were only altered by the higher dose (500 µM) of the drug. By contrast, in the presence of EPA/DHA (50/50 µM), all parameters showed a strongly improved response and the lowest UDCA dosage (50 µM) provided equal or better benefits than the highest dose used alone. For example, the combination EPA/DHA + UDCA 50 µM caused comparable down-regulation of the CYP7A1 gene expression and of the BA-induced caspase 3 activity as observed with UDCA 500 µM. In conclusion, these results suggest that the addition of n-3 PUFAs to UDCA may improve the response to the drug, and that such a pharmaco-nutraceutical approach could be used in clinic to open the narrow therapeutic dose of UDCA in cholestatic liver diseases.
Biliary obstruction, a severe cholestatic condition, results in a huge accumulation of toxic bile acids (BA) in the liver. Glucuronidation, a conjugation reaction, is thought to protect the liver by ...both reducing hepatic BA toxicity and increasing their urinary elimination. The present study evaluates the contribution of each process in the overall BA detoxification by glucuronidation. Glucuronide (G), glycine, taurine conjugates, and unconjugated BAs were quantified in pre- and post-biliary stenting urine samples from 12 patients with biliary obstruction, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The same LC-MS/MS procedure was used to quantify intra- and extracellular BA-G in Hepatoma HepG2 cells. Bile acid-induced toxicity in HepG2 cells was evaluated using MTS reduction, caspase-3 and flow cytometry assays. When compared to post-treatment samples, pre-stenting urines were enriched in glucuronide-, taurine- and glycine-conjugated BAs. Biliary stenting increased the relative BA-G abundance in the urinary BA pool, and reduced the proportion of taurine- and glycine-conjugates. Lithocholic, deoxycholic and chenodeoxycholic acids were the most cytotoxic and pro-apoptotic/necrotic BAs for HepG2 cells. Other species, such as the cholic, hyocholic and hyodeoxycholic acids were nontoxic. All BA-G assayed were less toxic and displayed lower pro-apoptotic/necrotic effects than their unconjugated precursors, even if they were able to penetrate into HepG2 cells. Under severe cholestatic conditions, urinary excretion favors the elimination of amidated BAs, while glucuronidation allows the conversion of cytotoxic BAs into nontoxic derivatives.
Bile acids (BA) are essential modulators of lipid, glucose, and cholesterol homeostasis, but they exert cytotoxic effects in the cholestatic liver. Glucuronidation, catalyzed by the ...UDP-glucuronosyltransferase (UGT) enzymes is a pharmacologically relevant BA detoxification process. The present study characterized the BA-conjugating activity of the little-studied human UGTs of subfamily 2A: UGT2A1, 2A2, and 2A3. Recombinant UGT2As, expressed in baculovirus-infected insect cells, were assayed for the glucuronidation of six major bile acids: chenodeoxycholic acid (CDCA), cholic acid (CA), lithocholic acid (LCA), deoxycholic acid (DCA), hyocholic acid (HCA) and hyodeoxycholic acid (HDCA). UGT2A3 exhibited detectable but very low activity with all the tested BA substrates. UGT2A1 was highly efficient in forming LCA-3 and LCA-24G, CDCA-24, DCA-24, HCA-24, and HDCA-24G, whereas UGT2A2 was the most active enzyme for CA-24G and CDCA-24G formation and also was able to generate HDCA-6G, HDCA-24G, LCA-24G, and HCA-24G. The Km values of UGT2A1 varied between 102.2 ± 14.3 µM and 2.4 ± 1.2 mM. With the exception of CA-24G, a low affinity substrate for UGT2A2, all the Km values for UGT2A2 were in the 100 to 400 µM range. We demonstrate the high reactivity of the human UGT2A1 and UGT2A2 for bile acid glucuronidation. The physiologic importance of these reactions to BA disposition remains, however, to be clarified in vivo.
Glucuronidation, catalyzed by UDP-glucuronosyltransferase UGT2B enzymes, is a major inactivating and elimination pathway for androgen hormones in humans. Whether Ugt2b enzymes from mice are also ...reactive with these hormones have never been investigated. The present study aimed at evaluating the capability of murine tissues and Ugt2b enzymes to glucuronidated androgens. The 7 murine Ugt2b (Ugt2b1, 2b5, 2b34, 2b35, 2b36, 2b37 and 2b38) enzymes were cloned and stably expressed into HEK293 cells. In vitro glucuronidation assays were performed with microsomal proteins or homogenates from mice tissues (liver, kidney, intestine, adipose, testis, prostate, epididymis, bulbo, seminal vesicle, mammary glands, uterus, and ovary) and from Ugt2b-HEK293 cells. Male and female livers, as well as male kidneys, are the major sites for androgen glucuronidation in mice. The male liver is highly efficient at glucuronidation of dihydrotestosterone (DHT) and testosterone and is enriched in Ugt2b1 and 2b5 enzymes. Androsterone and 3α-Diol are conjugated in the male kidney through an Ugt2b37-dependent process. Interestingly, castration partially abolished hepatic Ugt2b1 expression and activity, while Ugt2b37 was totally repressed. DHT injection partially corrected these changes. In conclusion, these observations revealed the substrate- and tissue-specific manner in which murine Ugt2b enzymes conjugate androgens. They also evidence how androgens modulate their own glucuronide conjugation in mice.
Cholestasis is characterized by the accumulation of toxic bile acids (BAs) in liver cells. The present study aimed to evaluate the effects of n-3 polyunsaturated fatty acids (n-3 PUFAs), such as ...docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, on BA homeostasis and toxicity in human cell models. The effects of EPA and/or DHA on the expression of genes involved in the maintenance of BA homeostasis were analyzed in human hepatoma (HepG2) and colon carcinoma (Caco-2) cells, as well as in primary culture of human intestinal (InEpC) and renal (RPTEC) cells. Extracellular BA species were quantified in culture media using LC-MS/MS. BA-induced toxicity was evaluated using caspase-3 and flow cytometry assays. Gene expression analyses of HepG2 cells reveal that n-3 PUFAs reduce the expression of genes involved in BA synthesis (CYP7A1, CYP27A1) and uptake (NTCP), while activating genes encoding metabolic enzymes (SULT2A1) and excretion transporters (MRP2, MRP3). N-3 PUFAs also generate a less toxic BA pool and prevent the BA-dependent activation of apoptosis in HepG2 cells. Conclusion. The present study reveals that n-3 PUFAs stimulate BA detoxification.
Bile acids subserve important physiological functions in the control of cholesterol homeostasis. Indeed, hepatic bile acid synthesis and biliary excretion constitute the main route for cholesterol ...removal from the human body. On the other hand, bile acids serve as natural detergents for the intestinal absorption of dietary cholesterol. However, due to their detergent properties, bile acids are inherently cytotoxic, and their cellular level may be tightly controlled to avoid pathological situations such as cholestasis. Recent investigations have illustrated the crucial roles that a series of ligand-activated transcription factors has in the control of hepatic bile acids synthesis, transport and metabolism. Thus, the lipid-activated nuclear receptors, farnesoid X-receptor (FXR), liver X-receptor (LXR), pregnane X-receptor (PXR) and peroxisome proliferator-activated receptor alpha (PPARα), modulate the expression and activity of genes controlling bile acid homeostasis in the liver. Several members of the UDP-glucuronosyltransferase (UGT) enzymes family are among the bile acid metabolizing enzymes regulated by these receptors. UGTs catalyze glucuronidation, a major phase II metabolic reaction, which converts hydrophobic bile acids into polar and urinary excretable metabolites. This article summarizes our recent observations on the regulation of bile acid conjugating UGTs upon pharmacological activation of lipid-activated receptors, with a particular interest for the role of PPARα and LXRα in controlling human UGT1A3 expression.
Biliary obstruction, a severe cholestatic complication, causes accumulation of toxic bile acids (BAs) in liver cells. Glucuronidation, catalyzed by UDP-glucuronosyltransferase (UGT) enzymes, ...detoxifies cholestatic BAs. Using liquid chromatography coupled to tandem mass spectrometry, 11 BA glucuronide (-G) species were quantified in prebiliary and postbiliary stenting serum and urine samples from 17 patients with biliary obstruction. Stenting caused glucuronide- and fluid-specific changes in BA-G levels and BA-G/BA metabolic ratios. In vitro glucuronidation assays with human liver and kidney microsomes revealed that even if renal enzymes generally displayed lower K M values, the two tissues shared similar glucuronidation capacities for BAs. By contrast, major differences between the two tissues were observed when four human BA-conjugating UGTs 1A3, 1A4, 2B4, and 2B7 were analyzed for mRNA and protein levels. Notably, the BA-24G producing UGT1A3 enzyme, abundant in the liver, was not detected in kidney microsomes. In conclusion, the circulating and urinary BA-G profiles are hugely impacted under severe cholestasis. The similar BA-glucuronidating abilities of hepatic and renal extracts suggest that both the liver and kidney may contribute to the urine BA-G pool.
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