Jocelyn Trottier

Tissue-specific, Inducible, and Hormonal Control of the Human UDP-Glucuronosyltransferase-1 (UGT1) Locus

The human UDP-glucuronosyltransferase 1 (UGT1) locus spans nearly 200 kb on chromosome 2 and encodes nine UGT1A proteins that play a prominent role in drug and xenobiotic metabolism. Transgenic UGT1 (Tg-UGT1) mice have been created, and it has been demonstrated that tissue-specific and xenobiotic receptor control of the UGT1A genes is influenced through circulating humoral factors. In Tg-UGT1 mice, the UGT1A proteins are differentially expressed in the liver and gastrointestinal tract. Gene expression profiles confirmed that all of the UGT1A genes can be targeted for regulation by the pregnane X receptor activator pregnenolone-16α-carbonitrile (PCN) or the Ah receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition, the selective induction of glucuronidation activity toward lamotrigine, ethinyl estradiol, chenodeoxycholic acid, and lithocholic acid by either PCN or TCDD in small intestine from Tg-UGT1 mice corresponded to expression of the locus in this tissue. Induction of UGT1A1 by PCN and TCDD is believed to be highly dependent upon glucocorticoids, because submicromolar concentrations of dexamethasone actively promote PCN and TCDD induction of UGT1A1 in Tg-UGT1 primary hepatocytes. The role of hormonal control of the UGT1 locus was further verified in pregnant and nursing Tg-UGT1 mice. In maternal 14-day post-conception Tg-UGT1mice, liver UGT1A1, UGT1A4, and UGT1A6 were induced, with the levels returning to near normal by birth. However, maternal liver UGT1A4 and UGT1A6 were dramatically elevated and maintained after birth, indicating that these proteins may play a critical role in maternal metabolism during lactation. With expression of the UGT1 locus confirmed in a variety of mouse tissues, these results suggested that the Tg-UGT1 mice will be a useful model to examine the regulatory and functional properties of human glucuronidation.

Expression of the human UGT1 locus in transgenic mice by 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY-14643) and implications on drug metabolism through peroxisome proliferator-activated receptor α activation

The UDP-glucuronosyltransferase (UGT) 1A genes in humans have been shown to be differentially regulated in a tissue-specific fashion. Transgenic mice carrying the human UGT1 locus (Tg-UGT1) were recently created, demonstrating that expression of the nine UGT1A genes closely resembles the patterns of expression observed in human tissues. In the present study, UGT1A1, UGT1A3, UGT1A4, and UGT1A6 have been identified as targets of the peroxisome proliferator-activated receptor (PPAR) α in human hepatocytes and Tg-UGT1 mice. Oral administration of the PPARα agonist 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (pirinixic acid, WY-14643) to Tg-UGT1 mice led to induction of these proteins in either the liver, gastrointestinal tract, or kidney. The levels of induced UGT1A3 gene transcripts in liver and UGT1A4 protein in small intestine correlated with induced lamotrigine glucuronidation activity in these tissues. With UGT1A3 previously identified as the major human enzyme involved in human C24-glucuronidation of lithocholic acid (LCA), the dramatic induction of liver UGT1A3 RNA in Tg-UGT1 mice was consistent with the formation of LCA-24G in plasma. Furthermore, PPAR-responsive elements (PPREs) were identified flanking the UGT1A1, UGT1A3, and UGT1A6 genes by a combination of site-directed mutagenesis, specific binding to PPARα and retinoic acid X receptor α, and functional response of the concatenated PPREs in HepG2 cells overexpressing PPARα. In conclusion, these results suggest that oral fibrate treatment in humans will induce the UGT1A family of proteins in the gastrointestinal tract and liver, influencing bile acid glucuronidation and first-pass metabolism of other drugs that are taken concurrently with hypolipidemic therapy.

Human UDP‐glucuronosyltransferase (UGT)1A3 enzyme conjugates chenodeoxycholic acid in the liver

Chenodeoxycholic acid (CDCA) is a liver‐formed detergent and plays an important role in the control of cholesterol homeostasis. During cholestasis, toxic bile acids (BA) accumulate in hepatocytes causing damage and consequent impairment of their function. Glucuronidation, a conjugation reaction catalyzed by UDP‐glucuronosyltransferase (UGT) enzymes, is considered an important metabolic pathway for hepatic BA. This study identifies the human UGT1A3 enzyme as the major enzyme responsible for the hepatic formation of the acyl CDCA‐24glucuronide (CDCA‐24G). Kinetic analyses revealed that human liver and UGT1A3 catalyze the formation of CDCA‐24G with similar Km values of 10.6 to 18.6 μmol/L, respectively. In addition, electrophoretic mobility shift assays and transient transfection experiments revealed that glucuronidation reduces the ability of CDCA to act as an activator of the nuclear farnesoid X‐receptor (FXR). Finally, we observed that treatment of human hepatocytes with fibrates increases the expression and activity of UGT1A3, whereas CDCA has no effect. In conclusion, UGT1A3 is the main UGT enzyme for the hepatic formation of CDCA‐24G and glucuronidation inhibits the ability of CDCA to act as an FXR activator. In vitro data also suggest that fibrates may favor the formation of bile acid glucuronides in cholestatic patients. (HEPATOLOGY 2006;44:1158–1170.)

The liver X‐receptor alpha controls hepatic expression of the human bile acid–glucuronidating UGT1A3 enzyme in human cells and transgenic mice

Glucuronidation, an important bile acid detoxification pathway, is catalyzed by enzymes belonging to the UDP‐glucuronosyltransferase (UGT) family. Among UGT enzymes, UGT1A3 is considered the major human enzyme for the hepatic C24‐glucuronidation of the primary chenodeoxycholic (CDCA) and secondary lithocholic (LCA) bile acids. We identify UGT1A3 as a positively regulated target gene of the oxysterol‐activated nuclear receptor liver X‐receptor alpha (LXRα). In human hepatic cells and human UGT1A transgenic mice, LXRα activators induce UGT1A3 mRNA levels and the formation of CDCA‐24glucuronide (24G) and LCA‐24G. Furthermore, a functional LXR response element (LXRE) was identified in the UGT1A3 promoter by site‐directed mutagenesis, electrophoretic mobility shift assays and chromatin immunoprecipitation experiment. In addition, LXRα is found to interact with the SRC‐1α and NCoR cofactors to regulate the UGT1A3 gene, but not with PGC‐1β. In conclusion, these observations establish LXRα as a crucial regulator of bile acid glucuronidation in humans and suggest that accumulation of oxysterols in hepatocytes during cholestasis favors bile acid detoxification as glucuronide conjugates. LXR agonists may be useful for stimulating both bile acid detoxification and cholesterol removal in cholestatic or hypercholesterolemic patients, respectively. (HEPATOLOGY 2006;44:368–378.)

Prospective evaluation of ursodeoxycholic acid withdrawal in patients with primary sclerosing cholangitis.

Ursodeoxycholic acid (UDCA) is no longer recommended for management of adult patients with primary sclerosing cholangitis (PSC). We undertook a prospective evaluation of UDCA withdrawal in a group of consecutive patients with PSC. Twenty six patients, all treated with UDCA (dose range: 10‐15 mg/kg/day) were included. Paired blood samples for liver biochemistry, bile acids, and fibroblast growth factor 19 (FGF19) were collected before UDCA withdrawal and 3 months later. Liquid chromatography/tandem mass spectrometry was used for quantification of 29 plasma bile acid metabolites. Pruritus and health‐related quality of life (HRQoL) were assessed with a 10‐point numeric rating scale, the Medical Outcomes Study Short Form‐36 (SF‐36), and PBC‐40 questionnaires. UDCA withdrawal resulted in a significant deterioration in liver biochemistry (increase of alkaline phosphatase of 75.6%; P < 0.0001; gamma‐glutamyl transpeptidase of 117.9%, P < 0.0001; bilirubin of 50.0%, P < 0.001; alanine aminotransferase of 63.9%, P < 0.005; and aspartate aminotransferase of 45.0%, P < 0.005) and increase of Mayo Risk Score for PSC (change from baseline of +0.5 point; P < 0.003). Bile acid analysis revealed a significant decrease in lithocholic acid and its derivatives after UDCA withdrawal, but no effect on concentrations of primary bile acids aside from an increased accumulation of their taurine conjugates. After UDCA removal cholestatic parameters, taurine species of cholic acid and chenodeoxycholic acid correlated with serum FGF19 levels. No significant effect on HRQoL after UDCA withdrawal was observed; however, 42% of patients reported a deterioration in their pruritus. Conclusion: At 3 months, discontinuation of UDCA in patients with PSC causes significant deterioration in liver biochemistry and influences concentrations of bile acid metabolites. A proportion of patients report increased pruritus, but other short‐term markers of quality of life are unaffected. (Hepatology 2014;60:931–940)

Metabolomic profiling of 17 bile acids in serum from patients with primary biliary cirrhosis and primary sclerosing cholangitis: A pilot study

BACKGROUND Primary biliary cirrhosis and primary sclerosing cholangitis are two cholestatic diseases characterised by hepatic accumulation of bile acids. AIMS This study compares serum bile acid levels in patients with primary biliary cirrhosis and primary sclerosing cholangitis and from age and sex-matched non cholestatic donors. METHODS Seventeen bile acids were quantified using liquid chromatography coupled to tandem mass spectrometry. Serum samples from cholestatic patients were compared with those of non-cholestatic donors. RESULTS The concentration of total bile acids, taurine and glycine conjugates of primary bile acids was elevated in both patients with primary biliary cirrhosis and primary sclerosing cholangitis when compared to non-cholestatic donors. Samples from primary sclerosing cholangitis patients displayed reduced levels of secondary acids, when compared to non cholestatic and primary biliary cirrhosis sera. The ratio of total glycine versus total taurine conjugates was reduced in patients with primary biliary cirrhosis, but not in primary sclerosing cholangitis. CONCLUSION The present study suggests that circulating bile acids are altered differentially in primary biliary cirrhosis and primary sclerosing cholangitis patients.

Profiling Circulating and Urinary Bile Acids in Patients with Biliary Obstruction before and after Biliary Stenting

Bile acids are considered as extremely toxic at the high concentrations reached during bile duct obstruction, but each acid displays variable cytotoxic properties. This study investigates how biliary obstruction and restoration of bile flow interferes with urinary and circulating levels of 17 common bile acids. Bile acids (conjugated and unconjugated) were quantified by liquid chromatography coupled with tandem mass spectrometry in serum and urine samples from 17 patients (8 men and 9 women) with biliary obstruction, before and after biliary stenting. Results were compared with serum concentrations measured in 40 age- and sex-paired control donors (20 men and 20 women). The total circulating bile acid concentration increases from 2.7 µM in control donors to 156.9 µM in untreated patients with biliary stenosis. Serum taurocholic and glycocholic acids exhibit 304- and 241-fold accumulations in patients with biliary obstruction compared to controls. The enrichment in chenodeoxycholic acid species reached a maximum of only 39-fold, while all secondary and 6α-hydroxylated species –except taurolithocholic acids – were either unchanged or significantly reduced. Stenting was efficient in restoring an almost normal circulating profile and in reducing urinary bile acids. Conclusion These results demonstrate that biliary obstruction affects differentially the circulating and/or urinary levels of the various bile acids. The observation that the most drastically affected acids correspond to the less toxic species supports the activation of self-protecting mechanisms aimed at limiting the inherent toxicity of bile acids in face of biliary obstruction.

Profile of serum bile acids in noncholestatic volunteers: Gender-related differences in response to fenofibrate

Fenofibrate belongs to the group of hypolipidemic fibrates that act as activators of the peroxisome proliferator–activated receptor‐α (PPARα), which is a regulator of bile acid synthesis, metabolism, and transport. The present study aimed at evaluating the effects of fenofibrate on the circulating bile acid profile in humans. A study population of 200 healthy individuals comprising both genders completed a 3‐week intervention with fenofibrate, and 17 bile acid species were measured in serum samples drawn before and after fenofibrate treatment. Fenofibrate caused significant reductions in levels of chenodeoxycholic (CDCA) (−26.4%), ursodeoxycholic (UDCA) (−30.5%), lithocholic (LCA) (−18.4%), deoxycholic (DCA) (−22.3%), and hyodeoxycholic (HDCA) (−19.2%) acids. A gender‐related difference was observed in the responses of various bile acids, and the total bile acid concentration was significantly reduced only in men (−18.6%), whereas it remained almost unchanged in women (+0.36%). This difference suggests that fenofibrate would be more efficient at reducing bile acid toxicity in men than in women in cholestatic liver diseases.

Lipid-activated transcription factors control bile acid glucuronidation

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.

Long noncoding RNA MEG3 induces cholestatic liver injury by interaction with PTBP1 to facilitate shp mRNA decay

Bile acids (BAs) play critical physiological functions in cholesterol homeostasis, and deregulation of BA metabolism causes cholestatic liver injury. The long noncoding RNA maternally expressed gene 3 (MEG3) was recently shown as a potential tumor suppressor; however, its basic hepatic function remains elusive. Using RNA pull‐down with biotin‐labeled sense or anti‐sense MEG 3RNA followed by mass spectrometry, we identified RNA‐binding protein polypyrimidine tract‐binding protein 1 (PTBP1) as a MEG3 interacting protein and validated their interaction by RNA immunoprecipitation (RIP). Bioinformatics analysis revealed putative binding sites for PTBP1 within the coding region (CDS) of small heterodimer partner (SHP), a key repressor of BA biosynthesis. Forced expression of MEG3 in hepatocellular carcinoma cells guided and facilitated PTBP1 binding to the Shp CDS, resulting in Shp mRNA decay. Transient overexpression of MEG3 RNA in vivo in mouse liver caused rapid Shp mRNA degradation and cholestatic liver injury, which was accompanied by the disruption of BA homeostasis, elevation of liver enzymes, as well as dysregulation of BA synthetic enzymes and metabolic genes. Interestingly, RNA sequencing coupled with quantitative PCR (qPCR) revealed a drastic induction of MEG3 RNA in Shp−/− liver. SHP inhibited MEG3 gene transcription by repressing cAMP response element‐binding protein (CREB) transactivation of the MEG3 promoter. In addition, the expression of MEG3 and PTBP1 was activated in human fibrotic and cirrhotic livers. Conclusion: MEG3 causes cholestasis by serving as a guide RNA scaffold to recruit PTBP1 to destabilize Shp mRNA. SHP in turn represses CREB‐mediated activation of MEG3 expression in a feedback‐regulatory fashion. (Hepatology 2017;65:604‐615).