The farnesoid X-receptor is an essential regulator of cholesterol homeostasis

To address the importance of the farnesoid X-receptor (FXR; NR1H4) for normal cholesterol homeostasis, we evaluated the major pathways of cholesterol metabolism in the FXR-deficient (-/-) mouse model. Compared with wild-type, FXR(-/-) mice have increased plasma high density lipoprotein (HDL) cholesterol and a markedly reduced rate of plasma HDL cholesterol ester clearance. Concomitantly, FXR(-/-) mice exhibit reduced expression of hepatic genes involved in reverse cholesterol transport, most notably, that for scavenger receptor BI. FXR(-/-) mice also have increased: (i) plasma non-HDL cholesterol and triglyceride levels, (ii) apolipoprotein B-containing lipoprotein synthesis, and (iii) intestinal cholesterol absorption. Surprisingly, biliary cholesterol elimination was increased in FXR(-/-) mice, despite decreased expression of hepatic genes thought to be involved in this process. These data demonstrate that FXR is a critical regulator of normal cholesterol metabolism and that genetic changes affecting FXR function have the potential to be pro-atherogenic.     

Liver-specific FGFR4 knockdown in mice on an HFD increases bile acid synthesis and improves hepatic steatosis

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease with increased risk in patients with metabolic syndrome. There are no FDA-approved treatments, but FXR agonists have shown promising results in clinical studies for NAFLD management. In addition to FXR, fibroblast growth factor receptor FGFR4 is a key mediator of hepatic bile acid synthesis. Using N-acetylgalactosamine–conjugated siRNA, we knocked down FGFR4 specifically in the liver of mice on chow or high-fat diet and in mouse primary hepatocytes to determine the role of FGFR4 in metabolic processes and hepatic steatosis. Liver-specific FGFR4 silencing increased bile acid production and lowered serum cholesterol. Additionally, we found that high-fat diet–induced liver steatosis and insulin resistance improved following FGFR4 knockdown. These improvements were associated with activation of the FXR-FGF15 axis in intestinal cells, but not in hepatocytes. We conclude that targeting FGFR4 in the liver to activate the intestinal FXR-FGF15 axis may be a promising strategy for the treatment of NAFLD and metabolic dysfunction.     

Identification of Novel Pathways That Control Farnesoid X Receptor-mediated Hypocholesterolemia

Farnesoid X receptor (FXR) plays important regulatory roles in bile acid, lipoprotein, and glucose homeostasis. Here, we have utilized Fxr^−/− mice and mice deficient in scavenger receptor class B type I (SR-BI), together with an FXR-specific agonist and adenovirus expressing hepatocyte nuclear factor 4α or constitutively active FXR, to identify the mechanisms by which activation of FXR results in hypocholesterolemia. We identify a novel pathway linking FXR to changes in hepatic p-JNK, hepatocyte nuclear factor 4α, and finally SR-BI. Importantly, we demonstrate that the FXR-dependent increase in SR-BI results in both hypocholesterolemia and an increase in reverse cholesterol transport, a process involving the transport of cholesterol from peripheral macrophages to the liver for excretion into the feces. In addition, we demonstrate that FXR activation also induces an SR-BI-independent increase in reverse cholesterol transport and reduces intestinal cholesterol absorption. Together, these data indicate that FXR is a promising therapeutic target for treatment of hypercholesterolemia and coronary heart disease.     

Secondary (iso)BAs cooperate with endogenous ligands to activate FXR under physiological and pathological conditions

IsoBAs, stereoisomers of primary and secondary BAs, are found in feces and plasma of human individuals. BA signaling via the nuclear receptor FXR is crucial for regulation of hepatic and intestinal physiology/pathophysiology. Aim: Investigate the ability of BA-stereoisomers to bind and modulate FXR under physiological/pathological conditions. Methods: Expression-profiling, luciferase-assays, fluorescence-based coactivator-association assays, administration of (iso)-BAs to WT and cholestatic mice. Results: Compared to CDCA/isoCDCA, administration of DCA/isoDCA, UDCA/isoUDCA only slightly increased mRNA expression of FXR target genes; the induction was more evident looking at pre-mRNAs. Notably, almost 50% of isoBAs were metabolized to 3-oxo-BAs within 4 h in cell-based assays, making it difficult to study their actions. FRET-based real-time monitoring of FXR activity revealed that isoCDCA>CDCA stimulated FXR, and isoDCA and isoUDCA allowed fully activated FXR to be re-stimulated by a second dose of GW4064. In vivo co-administration of a single dose of isoBAs followed by GW4064 cooperatively activated FXR, as did feeding of UDCA in a background of endogenous FXR ligands. However, in animals with biliary obstruction and concomitant loss of intestinal BAs, UDCA was unable to increase intestinal Fgf15. In contrast, mice with an impaired enterohepatic circulation of BAs (Asbt?/?, Ostα?/?), administration of UDCA was still able to induce ileal Fgf15 and repress hepatic BA-synthesis, arguing that UDCA is only effective in the presence of endogenous FXR ligands. Conclusion: Secondary (iso)BAs cooperatively activate FXR in the presence of endogenous BAs, which is important to consider in diseases linked to disturbances in BA enterohepatic cycling.     

The cholesterol-raising factor from coffee beans, cafestol, as an agonist ligand for the farnesoid and pregnane X receptors

Cafestol, a diterpene present in unfiltered coffee brews such as Scandinavian boiled, Turkish, and cafetière coffee, is the most potent cholesterol-elevating compound known in the human diet. Several genes involved in cholesterol homeostasis have previously been shown to be targets of cafestol, including cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid biosynthesis. We have examined the mechanism by which cafestol elevates serum lipid levels. Changes in several lipid parameters were observed in cafestol-treated APOE3Leiden mice, including a significant increase in serum triglyceride levels. Microarray analysis of these mice identified alterations in hepatic expression of genes involved in lipid metabolism and detoxification, many of which are regulated by the nuclear hormone receptors farnesoid X receptor (FXR) and pregnane X receptor (PXR). Further studies demonstrate that cafestol is an agonist ligand for FXR and PXR, and that cafestol down-regulates expression of the bile acid homeostatic genes CYP7A1, sterol 12alpha-hydroxylase, and Na(+)-taurocholate cotransporting polypeptide in the liver of wild-type but not FXR null mice. Cafestol did not affect genes known to be up-regulated by FXR in the liver of wild-type mice, but did increase expression of the positive FXR-target genes intestinal bile acid-binding protein and fibroblast growth factor 15 (FGF15) in the intestine. Because FGF15 has recently been shown to function in an enterohepatic regulatory pathway to repress liver expression of bile acid homeostatic genes, its direct induction in the gut may account for indirect effects of cafestol on liver gene expression. PXR-dependent gene regulation of cytochrome P450 3A11 and other targets by cafestol was also only seen in the intestine. Using a double FXR/PXR knockout mouse model, we found that both receptors contribute to the cafestol-dependent induction of intestinal FGF15 gene expression. In conclusion, cafestol acts as an agonist ligand for both FXR and PXR, and this may contribute to its impact on cholesterol homeostasis.     

Polyunsaturated fatty acids are FXR ligands and differentially regulate expression of FXR targets

Polyunsaturated fatty acids (PUFAs) have been previously reported as agonists of peroxisome proliferatoractivated receptor and antagonists of the liver X receptor. The activities on these two nuclear receptors have been attributed to their beneficial effects such as improvement of dyslipidemia and insulin sensitivity and decrease of hepatic lipogenesis. Here we report that PUFAs are ligands of farnesoid X receptor (FXR), a nuclear receptor for bile acids. In a conventional FXR binding assay, arachidonic acid (AA, 20:4), docosahexaenoic acid (DA, 22:6), and linolenic acid (LA, 18:3) had an affinity of 2.6, 1.5, and 3.5 microM, respectively. In a cell-free coactivator association assay, AA, DA, and LA decreased FXR agonist-induced FXR activation with IC(50)s ranging from 0.9 to 4.7 microM. In HepG2 cells, PUFAs regulated the expression of two FXR targets, BSEP and kininogen, in an opposite fashion, although both genes were transactivated by FXR. All three PUFAs dose-dependently enhanced FXR agonist-induced BSEP expression but decreased FXR agonist-induced human kininogen mRNA. Saturated fatty acids such as stearic acid (SA, 18:0) and palmitic acid (PA, 16:0) did not bind to FXR and did not change BSEP or kininogen expression. The pattern of BSEP and kininogen regulation by PUFAs is closely similar to that of the guggulsterone, previously reported as a selective bile acid receptor modulator. Our results suggest that PUFAs may belong to the same class of FXR ligands as guggulsterone, and that the selective regulation of FXR targets may contribute to the beneficial effects of PUFAs in lipid metabolism.     

Hepatic deletion of SIRT1 decreases hepatocyte nuclear factor 1α/farnesoid X receptor signaling and induces formation of cholesterol gallstones in mice

SIRT1, a highly conserved NAD(+)-dependent protein deacetylase, is a key metabolic sensor that directly links nutrient signals to animal metabolic homeostasis. Although SIRT1 has been implicated in a number of hepatic metabolic processes, the mechanisms by which hepatic SIRT1 modulates bile acid metabolism are still not well understood. Here we report that deletion of hepatic SIRT1 reduces the expression of farnesoid X receptor (FXR), a nuclear receptor that regulates bile acid homeostasis. We provide evidence that SIRT1 regulates the expression of FXR through hepatocyte nuclear factor 1α (HNF1α). SIRT1 deficiency in hepatocytes leads to decreased binding of HNF1α to the FXR promoter. Furthermore, we show that hepatocyte-specific deletion of SIRT1 leads to derangements in bile acid metabolism, predisposing the mice to development of cholesterol gallstones on a lithogenic diet. Taken together, our findings indicate that SIRT1 plays a vital role in the regulation of hepatic bile acid homeostasis through the HNF1α/FXR signaling pathway.     

FXR signaling in metabolic disease

Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily, has been shown to be important in controlling numerous metabolic pathways; these include roles in maintaining bile acid, lipid and glucose homeostasis, in preventing intestinal bacterial infection and gallstone formation and in modulating liver regeneration and tumorigenesis. The accumulating data suggest that FXR may be a pharmaceutical target for the treatment of certain metabolic diseases.     

The farnesoid X receptor modulates hepatic carbohydrate metabolism during the fasting-refeeding transition

The liver plays a central role in the control of blood glucose homeostasis by maintaining a balance between glucose production and utilization. The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor. Hepatic FXR expression is regulated by glucose and insulin. Here we identify a role for FXR in the control of hepatic carbohydrate metabolism. When submitted to a controlled fasting-refeeding schedule, FXR(-/-) mice displayed an accelerated response to high carbohydrate refeeding with an accelerated induction of glycolytic and lipogenic genes and a more pronounced repression of gluconeogenic genes. Plasma insulin and glucose levels were lower in FXR(-/-) mice upon refeeding the high-carbohydrate diet. These alterations were paralleled by decreased hepatic glycogen content. Hepatic insulin sensitivity was unchanged in FXR(-/-) mice. Treatment of isolated primary hepatocytes with a synthetic FXR agonist attenuated glucose-induced mRNA expression as well as promoter activity of L-type pyruvate kinase, acetyl-CoA carboxylase 1, and Spot14. Moreover, activated FXR interfered negatively with the carbohydrate response elements regions. These results identify a novel role for FXR as a modulator of hepatic carbohydrate metabolism.     

Bile acids and their nuclear receptor FXR: Relevance for hepatobiliary and gastrointestinal disease

The nuclear receptor Farnesoid X Receptor (FXR) critically regulates nascent bile formation and bile acid enterohepatic circulation. Bile acids and FXR play a pivotal role in regulating hepatic inflammation and regeneration as well as in regulating extent of inflammatory responses, barrier function and prevention of bacterial translocation in the intestinal tract. Recent evidence suggests, that the bile acid–FXR interaction is involved in the pathophysiology of a wide range of diseases of the liver, biliary and gastrointestinal tract, such as cholestatic and inflammatory liver diseases and hepatocellular carcinoma, inflammatory bowel disease and inflammation-associated cancer of the colon and esophagus. In this review we discuss current knowledge of the role the bile acid–FXR interaction has in (patho)physiology of the liver, biliary and gastrointestinal tract, and proposed underlying mechanisms, based on in vitro data and experimental animal models. Given the availability of highly potent synthetic FXR agonists, we focus particularly on potential relevance for human disease.     

Abolished synthesis of cholic acid reduces atherosclerotic development in apolipoprotein E knockout mice

To investigate the effects of abolished cholic acid (CA) synthesis in the ApoE knockout model [apolipoprotein E (apoE) KO],a double-knockout (DKO) mouse model was created by crossbreeding Cyp8b1 knockout mice (Cyp8b1 KO), unable to synthesize the primary bile acid CA, with apoE KO mice. After 5 months of cholesterol feeding, the development of atherosclerotic plaques in the proximal aorta was 50% less in the DKO mice compared with the apoE KO mice. This effect was associated with reduced intestinal cholesterol absorption, decreased levels of apoB-containing lipoproteins in the plasma, enhanced bile acid synthesis, reduced hepatic cholesteryl esters, and decreased hepatic activity of ACAT2. The upregulation of Cyp7a1 in DKO mice seemed primarily caused by reduced expression of the intestinal peptide FGF15. Treatment of DKO mice with the farnesoid X receptor (FXR) agonist GW4064 did not alter the intestinal cholesterol absorption, suggesting that the action of CA in this process is confined mainly to formation of intraluminal micelles and less to its ability to activate the nuclear receptor FXR. Inhibition of CA synthesis may offer a therapeutic strategy for the treatment of hyperlipidemic conditions that lead to atherosclerosis.