John Y. L. Chiang

Bile acids: regulation of synthesis

Bile acids are physiological detergents that generate bile flow and facilitate intestinal absorption and transport of lipids, nutrients, and vitamins. Bile acids also are signaling molecules and inflammatory agents that rapidly activate nuclear receptors and cell signaling pathways that regulate lipid, glucose, and energy metabolism. The enterohepatic circulation of bile acids exerts important physiological functions not only in feedback inhibition of bile acid synthesis but also in control of whole-body lipid homeostasis. In the liver, bile acids activate a nuclear receptor, farnesoid X receptor (FXR), that induces an atypical nuclear receptor small heterodimer partner, which subsequently inhibits nuclear receptors, liver-related homolog-1, and hepatocyte nuclear factor 4&agr; and results in inhibiting transcription of the critical regulatory gene in bile acid synthesis, cholesterol 7&agr;-hydroxylase (CYP7A1). In the intestine, FXR induces an intestinal hormone, fibroblast growth factor 15 (FGF15; or FGF19 in human), which activates hepatic FGF receptor 4 (FGFR4) signaling to inhibit bile acid synthesis. However, the mechanism by which FXR/FGF19/FGFR4 signaling inhibits CYP7A1 remains unknown. Bile acids are able to induce FGF19 in human hepatocytes, and the FGF19 autocrine pathway may exist in the human livers. Bile acids and bile acid receptors are therapeutic targets for development of drugs for treatment of cholestatic liver diseases, fatty liver diseases, diabetes, obesity, and metabolic syndrome.

Regulation of cholesterol 7α-hydroxylase gene (CYP7A1) transcription by the liver orphan receptor (LXRα)

Abstract The cholesterol 7α-hydroxylase gene (CYP7A1) plays an important role in regulation of bile acid biosynthesis and cholesterol homeostasis. Oxysterol receptor, LXR, stimulates, whereas the bile acid receptor, FXR, inhibits CYP7A1 transcription. The goal of this study was to investigate the role of LXRα on the regulation of rat, human and hamster CYP7A1 transcription in its native promoter and cellular context. Cotransfection with LXRα and RXRα expression plasmids strongly stimulated rat CYP7A1/luciferase reporter activity in HepG2 cells and oxysterol was not required. However, LXRα had much less effect on hamster and no significant effect on human CYP7A1 promoter activity in HepG2 cells. In Chinese hamster ovary cells, cotransfection with LXRα stimulated reporter activity by less than 2-fold and addition of 22(R)-hydroxycholesterol caused a small but significant stimulation of rat, human and hamster CYP7A1 promoter activity. At least two direct repeats of AGGTCA-like sequences with 4-base spacing (DR4) and five-base spacing (DR5), in previously identified bile acid response elements of the rat CYP7A1 were able to bind LXRα/RXRα and confer LXRα stimulation. However, LXRα did not bind to the corresponding sequences of the human gene and bound weakly to hamster and mouse DR4 sequences. Therefore, rats and mice have the unusual capacity to convert cholesterol to bile acids by LXRα-mediated stimulation of CYP7A1 transcription, whereas other species do not respond to cholesterol and develop hypercholesterolemia on a diet high in cholesterol.

REGULATION OF BILE ACID SYNTHESIS

Bile acids are important physiological agents required for disposal of cholesterol and absorption of vitamins and fats. Bile acids are synthesized from cholesterol in the liver. Enterohepatic circulation of bile acids is very efficient and plays an important physiological role in lipid absorption and secretion, and regulation of bile acid biosynthesis and cholesterol homeostasis. Conversion of cholesterol to bile acids requires 15 different enzymatic steps. Four cytochrome P450 enzymes play important roles in bile acid biosynthesis. The classic bile acid biosynthesis pathway starts with modification of the sterol ring and followed by side chain cleavage reactions to synthesize cholic acid (CA) and chenodeoxycholic acid (CDCA), the primary bile acids in most species. The first and rate-limiting enzyme in this pathway is cholesterol 7alpha -hydroxylase, a microsomal cytochrome P450, CYP7A. Another microsomal cytochrome P450 sterol 12alpha-hydroxylase (CYP12) is required for the synthesis of cholic acid. Mitochondrial cytochrome P450 sterol 27-hydroxylase (CYP27) catalyzes sterol side chain oxidation to convert C27 sterol to C24 bile acids. An alternative bile acid biosynthesis pathway (acidic) has been known for sometime but only recently has attracted much attention. In this pathway, side chain oxidation precedes modification of the sterol ring. Mitochondrial sterol 27-hydroxylase (CYP27) catalyzes the first reaction and followed by 7alpha-hydroxylation catalyzed by a microsomal oxysterol 7alpha-hydroxylase (CYP7B). Recent advances in purification and cloning of these major enzymes in the pathways have led to better understanding the molecular basis of regulation of bile acid synthesis and physiological role of the alternative pathways.

Bile Acid Signaling in Metabolic Disease and Drug Therapy

Bile acids are the end products of cholesterol catabolism. Hepatic bile acid synthesis accounts for a major fraction of daily cholesterol turnover in humans. Biliary secretion of bile acids generates bile flow and facilitates hepatobiliary secretion of lipids, lipophilic metabolites, and xenobiotics. In the intestine, bile acids are essential for the absorption, transport, and metabolism of dietary fats and lipid-soluble vitamins. Extensive research in the last 2 decades has unveiled new functions of bile acids as signaling molecules and metabolic integrators. The bile acid–activated nuclear receptors farnesoid X receptor, pregnane X receptor, constitutive androstane receptor, vitamin D receptor, and G protein–coupled bile acid receptor play critical roles in the regulation of lipid, glucose, and energy metabolism, inflammation, and drug metabolism and detoxification. Bile acid synthesis exhibits a strong diurnal rhythm, which is entrained by fasting and refeeding as well as nutrient status and plays an important role for maintaining metabolic homeostasis. Recent research revealed an interaction of liver bile acids and gut microbiota in the regulation of liver metabolism. Circadian disturbance and altered gut microbiota contribute to the pathogenesis of liver diseases, inflammatory bowel diseases, nonalcoholic fatty liver disease, diabetes, and obesity. Bile acids and their derivatives are potential therapeutic agents for treating metabolic diseases of the liver.

Bile Acid Metabolism and Signaling

Bile acids are important physiological agents for intestinal nutrient absorption and biliary secretion of lipids, toxic metabolites, and xenobiotics. Bile acids also are signaling molecules and metabolic regulators that activate nuclear receptors and G protein-coupled receptor (GPCR) signaling to regulate hepatic lipid, glucose, and energy homeostasis and maintain metabolic homeostasis. Conversion of cholesterol to bile acids is critical for maintaining cholesterol homeostasis and preventing accumulation of cholesterol, triglycerides, and toxic metabolites, and injury in the liver and other organs. Enterohepatic circulation of bile acids from the liver to intestine and back to the liver plays a central role in nutrient absorption and distribution, and metabolic regulation and homeostasis. This physiological process is regulated by a complex membrane transport system in the liver and intestine regulated by nuclear receptors. Toxic bile acids may cause inflammation, apoptosis, and cell death. On the other hand, bile acid-activated nuclear and GPCR signaling protects against inflammation in liver, intestine, and macrophages. Disorders in bile acid metabolism cause cholestatic liver diseases, dyslipidemia, fatty liver diseases, cardiovascular diseases, and diabetes. Bile acids, bile acid derivatives, and bile acid sequestrants are therapeutic agents for treating chronic liver diseases, obesity, and diabetes in humans.

Mechanism of tissue-specific farnesoid X receptor in suppressing the expression of genes in bile-acid synthesis in mice†

Activation of farnesoid X receptor (Fxr, Nr1h4) is a major mechanism in suppressing bile‐acid synthesis by reducing the expression levels of genes encoding key bile‐acid synthetic enzymes (e.g., cytochrome P450 [CYP]7A1/Cyp7a1 and CYP8B1/Cyp8b1). FXR‐mediated induction of hepatic small heterodimer partner (SHP/Shp, Nr0b2) and intestinal fibroblast growth factor 15 (Fgf15; FGF19 in humans) has been shown to be responsible for this suppression. However, the exact contribution of Shp/Fgf15 to this suppression, and the associated cell‐signaling pathway, is unclear. By using novel genetically modified mice, the current study showed that the intestinal Fxr/Fgf15 pathway was critical for suppressing both Cyp7a1 and Cyp8b1 gene expression, but the liver Fxr/Shp pathway was important for suppressing Cyp8b1 gene expression and had a minor role in suppressing Cyp7a1 gene expression. Furthermore, in vivo administration of Fgf15 protein to mice led to a strong activation of extracellular signal‐related kinase (ERK) and, to a smaller degree, Jun N‐terminal kinase (JNK) in the liver. In addition, deficiency of either the ERK or JNK pathway in mouse livers reduced the basal, but not the Fgf15‐mediated, suppression of Cyp7a1 and Cyp8b1 gene expression. However, deficiency of both ERK and JNK pathways prevented Fgf15‐mediated suppression of Cyp7a1 and Cyp8b1 gene expression. Conclusion: The current study clearly elucidates the underlying molecular mechanism of hepatic versus intestinal Fxr in regulating the expression of genes critical for bile‐acid synthesis and hydrophobicity in the liver. (HEPATOLOGY 2012;56:1034–1043)

Effect of thyroid hormone on hepatic cholesterol 7α hydroxylase, LDL receptor, HMG-CoA reductase, farnesyl pyrophosphate synthetase and apolipoprotein A-I mRNA levels in hypophysectomized rats

The effects of thyroid hormone on cholesterol 7 alpha hydroxylase, LDL receptor, HMG-CoA reductase, apo A-I and farnesyl pyrophosphate synthetase hepatic mRNA levels were investigated in hypophysectomized rats. Of these mRNAs cholesterol 7 alpha hydroxylase responded the most rapidly and required the lowest dose of T3. Maximal mRNA levels were reached one hr after T3 administration and required 10 micrograms/100g of body weight. These results suggest that the hypocholesterolemic effect of thyroid hormone may be mediated by a primary effect on cholesterol 7 alpha hydroxylase gene expression.

Bile acids and cytokines inhibit the human cholesterol 7α-hydroxylase gene via the JNK/c-jun pathway in human liver cells

Cholesterol 7α‐hydroxylase (CYP7A1) of the bile acid biosynthesis pathway is suppressed by bile acids and inflammatory cytokines. Bile acids are known to induce inflammatory cytokines to activate the mitogen‐activated protein kinase/c‐Jun N‐terminal kinase (JNK) signaling pathway that inhibits CYP7A1 gene transcription. c‐Jun has been postulated to mediate bile acid inhibition of CYP7A1. However, the c‐Jun target involved in the regulation of CYP7A1 is unknown. Human primary hepatocytes and HepG2 cells were used as models to study chenodeoxycholic acid (CDCA) and interleukin‐1β (IL‐1β) regulation of human CYP7A1 gene expression via real‐time polymerase chain reaction, reporter assays, co‐immunoprecipitation and chromatin immunocipitation (ChIP) assays. IL‐1β and CDCA reduced CYP7A1 but induced c‐Jun messenger RNA expression in human primary hepatocytes. IL‐1β inhibited human CYP7A1 reporter activity via the HNF4α binding site. A JNK‐specific inhibitor blocked the inhibitory effect of IL‐1β on HNF4α expression and CYP7A1 reporter activity. c‐Jun inhibited HNF4α and PPARγ coactivator‐1α (PGC‐1α) coactivation of CYP7A1 reporter activity, whereas a dominant negative c‐Jun did not. Co‐immunoprecipitation and ChIP assays revealed that IL‐1β and CDCA reduced HNF4α bound to the CYP7A1 chromatin, and that c‐Jun interacted with HNF4α and blocked HNF4α recruitment of PGC‐1α to the CYP7A1 chromatin. In conclusion, IL‐1β and CDCA inhibit HNF4α but induce c‐Jun, which in turn blocks HNF4α recruitment of PGC‐1α to the CYP7A1 chromatin and results in inhibition of CYP7A1 gene transcription. The JNK/c‐Jun signaling pathway inhibits bile acid synthesis and protects hepatocytes against the toxic effect of inflammatory agents. (HEPATOLOGY 2006;43:1202–1210.)

Glucose and Insulin Induction of Bile Acid Synthesis MECHANISMS AND IMPLICATION IN DIABETES AND OBESITY

Background: Bile acid synthesis plays an important role in nutrient absorption and maintaining metabolic homeostasis under normal physiology. Results: Glucose induces cholesterol 7α-hydroxylase activity and postprandial bile acid synthesis via insulin signaling and epigenetic mechanisms. Conclusion: Glucose and insulin are major postprandial factors that stimulate bile acid synthesis to maintain hepatic metabolic homeostasis. Significance: Nutrient regulation of bile acid synthesis is impaired in diabetes and obesity. Bile acids facilitate postprandial absorption of nutrients. Bile acids also activate the farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5 and play a major role in regulating lipid, glucose, and energy metabolism. Transgenic expression of cholesterol 7α-hydroxylase (CYP7A1) prevented high fat diet-induced diabetes and obesity in mice. In this study, we investigated the nutrient effects on bile acid synthesis. Refeeding of a chow diet to fasted mice increased CYP7A1 expression, bile acid pool size, and serum bile acids in wild type and humanized CYP7A1-transgenic mice. Chromatin immunoprecipitation assays showed that glucose increased histone acetylation and decreased histone methylation on the CYP7A1 gene promoter. Refeeding also induced CYP7A1 in fxr-deficient mice, indicating that FXR signaling did not play a role in postprandial regulation of bile acid synthesis. In streptozocin-induced type I diabetic mice and genetically obese type II diabetic ob/ob mice, hyperglycemia increased histone acetylation status on the CYP7A1 gene promoter, leading to elevated basal Cyp7a1 expression and an enlarged bile acid pool with altered bile acid composition. However, refeeding did not further increase CYP7A1 expression in diabetic mice. In summary, this study demonstrates that glucose and insulin are major postprandial factors that induce CYP7A1 gene expression and bile acid synthesis. Glucose induces CYP7A1 gene expression mainly by epigenetic mechanisms. In diabetic mice, CYP7A1 chromatin is hyperacetylated, and fasting to refeeding response is impaired and may exacerbate metabolic disorders in diabetes.

Overexpression of Cholesterol 7α-hydroxylase promotes hepatic bile acid synthesis and secretion and maintains cholesterol homeostasis

We reported previously that mice overexpressing cytochrome P450 7a1 (Cyp7a1; Cyp7a1‐tg mice) are protected against high fat diet–induced hypercholesterolemia, obesity, and insulin resistance. Here, we investigated the underlying mechanism of bile acid signaling in maintaining cholesterol homeostasis in Cyp7a1‐tg mice. Cyp7a1‐tg mice had two‐fold higher Cyp7a1 activity and bile acid pool than did wild‐type mice. Gallbladder bile acid composition changed from predominantly cholic acid (57%) in wild‐type to chenodeoxycholic acid (54%) in Cyp7a1‐tg mice. Cyp7a1‐tg mice had higher biliary and fecal cholesterol and bile acid secretion rates than did wild‐type mice. Surprisingly, hepatic de novo cholesterol synthesis was markedly induced in Cyp7a1‐tg mice but intestine fractional cholesterol absorption in Cyp7a1‐tg mice remained the same as wild‐type mice despite the presence of increased intestine bile acids. Interestingly, hepatic but not intestinal expression of several cholesterol (adenosine triphosphate–binding cassette G5/G8 [ABCG5/G8], scavenger receptor class B, member 1) and bile acid (ABCB11) transporters were significantly induced in Cyp7a1‐tg mice. Treatment of mouse or human hepatocytes with a farnesoid X receptor (FXR) agonist GW4064 or bile acids induced hepatic Abcg5/g8 expression. A functional FXR binding site was identified in the Abcg5 gene promoter. Study of tissue‐specific Fxr knockout mice demonstrated that loss of the Fxr gene in the liver attenuated bile acid induction of hepatic Abcg5/g8 and gallbladder cholesterol content, suggesting a role of FXR in the regulation of cholesterol transport. Conclusion: This study revealed a new mechanism by which increased Cyp7a1 activity expands the hydrophobic bile acid pool, stimulating hepatic cholesterol synthesis and biliary cholesterol secretion without increasing intestinal cholesterol absorption. This study demonstrated that Cyp7a1 plays a critical role in maintaining cholesterol homeostasis and underscores the importance of bile acid signaling in regulating overall cholesterol homeostasis. (HEPATOLOGY 2011)