Roles of FGF19 in liver metabolism.

Fibroblast growth factor 19 (FGF19) is an ileum-derived postprandial enterokine that governs bile acid and nutrient metabolism. Synthesis of FGF19 is up-regulated by bile acids and, conversely, bile acid synthesis is down-regulated by FGF19. FGF19 also controls gallbladder volume. FGF19 has been shown to have profound effects on glucose and lipid metabolism. Recent studies have described FGF19 as a postprandial regulator of hepatic glucose and protein metabolism. Like insulin, FGF19 induces protein and glycogen synthesis and suppresses gluconeogenesis in liver. However, unlike insulin, FGF19 does not stimulate lipogenesis. A key difference between FGF19 and insulin lies in their use of different cellular signaling pathways. The beneficial effects of FGF19 on liver metabolism raise the question of whether FGF19 and its variants can be used as therapeutic agents in the treatment of diabetes.

Liver X receptors and cholesterol homoeostasis: spotlight on the adrenal gland.

The LXRs (liver X receptors) (LXRalpha and LXRbeta) are nuclear hormone receptors that are activated by oxysterols, endogenous oxidative metabolites of cholesterol. These receptors regulate an integrated network of genes that control whole body cholesterol and lipid homoeostasis. A brief overview of the mechanism of this regulation by LXRs in the liver, macrophage and intestine will be outlined, followed by data from our recent work demonstrating that LXRalpha is crucial in maintaining adrenal cholesterol homoeostasis. In the adrenal gland, oxysterols are formed as intermediates in the conversion of cholesterol into steroid hormones and can act as endogenous activators of LXR. We have found using both gain- and loss-of-function models that LXR acts to maintain free cholesterol below toxic levels in the adrenal gland, through the co-ordinated regulation of genes involved in cholesterol efflux [ABCA1 (ATP-binding-cassette transporter A1)], storage (sterol-regulatory-element-binding protein-1c and apolipoprotein E) and metabolism to steroid hormones (steroidogenic acute regulatory protein). Furthermore, we show that under chronic dietary stress, the adrenal glands of LXR-null mice (and not wild-type mice) accumulate free cholesterol. These results support the role of LXR as a global regulator of cholesterol homoeostasis, where LXR provides a safety valve to limit free cholesterol in tissues experiencing high cholesterol flux.

Nuclear oxysterol receptors, LXRs, are involved in the maintenance of mouse caput epididymidis structure and functions.

In this study we looked at the epididymides and spermatozoa of mice knocked-out for nuclear oxysterol receptors (LXR). We have shown that LXR-deficient mice exhibited upon ageing a severe disruption of their caput epididymides associated with abnormal accumulation of neutral lipids. The epididymis defaults were correlated with sperm head fragility and infertility. In agreement with the observed caput defect in transgenic animals in which both LXRalpha and LXRbeta isoforms were disrupted, we have shown here that both receptors are expressed in caput and cauda epididymides regions. LXRbeta was predominantly expressed throughout the mouse epididymis while the expression of LXRalpha was weaker. In addition, the expression of selected genes that can be considered as markers of adult epididymis function was monitored via Northern blots in the different single and double LXR-deficient backgrounds. Altogether, the data presented here suggest that LXR receptors are important actors in epididymis function.

PPAR alpha is necessary for the lipopenic action of hyperleptinemia on white adipose and liver tissue.

Adenovirus-induced hyperleptinemia causes rapid disappearance of body fat in normal rats, presumably by up-regulating fatty acid oxidation within white adipocytes. To determine the role of peroxisomal proliferation-activated receptor (PPAR)alpha expression, which was increased during the rapid loss of fat, we infused adenovirus-leptin into PPAR alpha(-/-) and PPAR alpha(+/+) mice. Despite similar degrees of hyperleptinemia and reduction in food intake, epididymal fat pad weight declined 55% in wild-type but only 6% in PPAR alpha(-/-) mice; liver triacylglycerol fell 39% in the wild-type group but was unchanged in PPAR(-/-) mice. Carnitine palmitoyl transferase-1 mRNA rose 52% in the wild-type mice but did not increase in PPAR alpha(-/-) mice. PPAR gamma coactivator-1 alpha rose 3-fold in the fat and 46% in the liver of wild-type mice but was unchanged in PPAR alpha(-/-) mice. Although AMP-activated protein kinase could not be implicated in the lipopenic actions of hyperleptinemia, acetyl CoA carboxylase protein was reduced in the liver of wild-type but not in PPAR alpha(-/-) mice. Thus, in PPAR alpha(-/-) mice, up-regulation of carnitine palmitoyl transferase-1 mRNA in fat, down-regulation of acetyl CoA carboxylase in liver, and up-regulation of PPAR gamma coactivator-1 alpha mRNA in both tissues are abolished, as is the reduction in their triacylglycerol content.

Nuclear receptors and lipid physiology: opening the X-files.

Cholesterol, fatty acids, fat-soluble vitamins, and other lipids present in our diets are not only nutritionally important but serve as precursors for ligands that bind to receptors in the nucleus. To become biologically active, these lipids must first be absorbed by the intestine and transformed by metabolic enzymes before they are delivered to their sites of action in the body. Ultimately, the lipids must be eliminated to maintain a normal physiological state. The need to coordinate this entire lipid-based metabolic signaling cascade raises important questions regarding the mechanisms that govern these pathways. Specifically, what is the nature of communication between these bioactive lipids and their receptors, binding proteins, transporters, and metabolizing enzymes that links them physiologically and speaks to a higher level of metabolic control? Some general principles that govern the actions of this class of bioactive lipids and their nuclear receptors are considered here, and the scheme that emerges reveals a complex molecular script at work.

Engineering orthogonal ligand-receptor pairs from "near drugs".

Cell-permeable small molecules are powerful tools for unraveling complex cellular pathways. We demonstrate that nuclear hormone receptors can be engineered through mutagenesis to create orthogonal ligand-receptor pairs to control transcription. Mutated residues in the retinoid X receptor (RXR) were chosen from structural analysis of RXR and the retinoic acid receptor (RAR) ligand binding domains. The potential ligands screened for activation of variant receptors are "near drugs"--compounds synthesized during structure-activity studies that are structurally similar to an approved drug yet inactive on the wild-type receptor. One variant, Q275C;I310M;F313I, is poorly activated by ligands for the wild-type receptor but is activated by a "near drug", fulfilling the criteria of an orthogonal ligand-receptor pair. These experiments demonstrate that nuclear hormone receptors are well suited to supply orthogonal ligand-receptor pairs for experimental biology, biotechnology, and gene therapy. Our findings also demonstrate the general principle that inactive compounds synthesized during drug discovery can be combined with mutant proteins to rapidly create new tools for controlling cellular processes.

Prospects for prevention and treatment of cancer with selective PPARgamma modulators (SPARMs).

Peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor and transcription factor that regulates the expression of many genes relevant to carcinogenesis, is now an important target for development of new drugs for the prevention and treatment of cancer. Deficient expression of PPARgamma can be a significant risk factor for carcinogenesis, although in some cases overexpression enhances carcinogenesis. Ligands for PPARgamma suppress breast carcinogenesis in experimental models and induce differentiation of human liposarcoma cells. By analogy to the selective estrogen receptor modulator (SERM) concept, it is suggested that selective PPARgamma modulators (SPARMs), designed to have desired effects on specific genes and target tissues without undesirable effects on others, will be clinically important in the future for chemoprevention and chemotherapy of cancer.

Regulation of lipoprotein lipase by the oxysterol receptors, LXRalpha and LXRbeta.

Lipoprotein lipase (LPL) is a key enzyme for lipoprotein metabolism and is responsible for hydrolysis of triglycerides in circulating lipoproteins, releasing free fatty acids to peripheral tissues. In liver, LPL is also believed to promote uptake of high density lipoprotein (HDL)-cholesterol and thereby facilitate reverse cholesterol transport. In this study we show that the Lpl gene is a direct target of the oxysterol liver X receptor, LXRalpha. Mice fed diets containing high cholesterol or an LXR-selective agonist exhibited a significant increase in LPL expression in the liver and macrophages, but not in other tissues (e.g. adipose and muscle). Studies in Lxr-deficient mice confirmed that this response was dependent more on the presence of LXRalpha than LXRbeta. Analysis of the Lpl gene revealed the presence of a functional DR4 LXR response element in the intronic region between exons 1 and 2. This response element directly binds rexinoid receptor (RXR)/LXR heterodimers and is sufficient for rexinoid- and LXR agonist-induced transcription of the Lpl gene. Together, these studies further distinguish the roles of LXRalpha and beta and support a growing body of evidence that LXRs function as key regulators of lipid metabolism and are anti-atherogenic.

[Regulation of lipid metabolism by the orphan nuclear receptors]. / Régulation du métabolisme des lipides par les récepteurs nucléaires orphelins.

Lipids (cholesterol and fatty acids) are essential nutriments and have a major impact on gene expression. Hence cholesterol intracellular concentration is precisely controlled by some complex mechanisms involving transcriptional regulations. The excess of cholesterol in cells is converted into oxysterols. These cholesterol metabolites are important signalisation molecules that modulate several transcription factors involved in cholesterol homeostasis. Schematically, regulation of cholesterol homeostasis is achieved by three different but complementary pathways: 1) endogeneous biosynthesis, which corresponds to the de novo synthesis of cholesterol and is controlled by sterol response element binding proteins (SREBPs); 2) the transport, intracellular absorption and esterification of the cholesterol; 3) the metabolic conversion into bile acids and steroid hormones. These three pathways are closely linked, however we will schematically detail the role of the orphan nuclear receptors on the modulation of these three levels of regulation. Phenotype analyses of knock-out or transgenic mice pointed out the respective role of the "enterohepatic" orphan nuclear receptors LXRalpha, LXRB, FXR, LRH-1, the nuclear receptor PPARalpha, and their heterodimeric partner RXR, as well as the peculiar receptor SHP. Complex feed-backs have thus been demonstrated. These transciptional regulations have several targets: the P450 cytochromes involved in the bile acid synthesis Cyp7a1 and Cyp8b1; the intestinal bile acid binding protein IBABP; the cholesteryl ester transfert protein CETP and phospholipid transfert protein PLTP, both involved in the HDL catabolism; the ABC cholesterol transporters ABCG1/ABC8 and ABCAI/ABCI. At last it seems that polyunsaturated fatty acids could activate LXRalpha transcription through its activation by PPARalpha. In the near future, the identification and study of new target genes by transcriptomic or proteomic analyses will allow a better understanding of lipid homeostasis in physiological as well as pathophysiological conditions.

The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp.

BACKGROUND AND AIMS: Hepatic bile acid homeostasis is regulated by negative feedback inhibition of genes involved in the uptake and synthesis of bile acids. Bile acids down-regulate the rate-limiting gene for bile acid synthesis, cholesterol 7alpha-hydroxylase (cyp7a), via bile acid receptor (fxr) activation of an inhibitory nuclear receptor, shp. We hypothesized that shp would also mediate negative feedback regulation of ntcp, the principal hepatic bile acid transporter. METHODS: Primary rat hepatocytes or transfected HepG2 and Cos cells were treated with retinoids with or without bile acids, and effects on bile acid transport and ntcp and shp gene expression and promoter activity were determined. Gel shift assays were performed using synthetic fxr, rxr, and rar proteins. RESULTS: Bile acid treatment of primary rat hepatocytes prevented retinoid activation of ntcp gene expression and function; this corresponded temporally with shp gene activation. Bile acid-mediated down-regulation occurred via fxr-dependent suppression of the ntcp RXR:RAR response element. Moreover, cotransfected shp directly inhibited retinoid activation of the ntcp promoter. CONCLUSIONS: These studies show negative feedback regulation of ntcp by bile acid-activated fxr via induction of shp. This novel regulatory pathway provides a means for coordinated down-regulation of bile acid import and synthesis, thereby protecting the hepatocyte from bile acid-mediated damage in cholestatic conditions.

Human bile salt export pump promoter is transactivated by the farnesoid X receptor/bile acid receptor.

The bile salt excretory pump (BSEP, ABCb11) is critical for ATP-dependent transport of bile acids across the hepatocyte canalicular membrane and for generation of bile acid-dependent bile secretion. Recent studies have demonstrated that the expression of this transporter is sensitive to the flux of bile acids through the hepatocyte, possibly at the level of transcription of the BSEP gene. To determine the mechanisms underlying the regulation of BSEP by bile acids, the promoter of the BSEP gene was cloned. The sequence of the promoter contained an inverted repeat (IR)-1 element (5'-GGGACA T TGATCCT-3') at base pairs -63/-50 consisting of two nuclear receptor half-sites organized as an inverted repeat and separated by a single nucleotide. This IR-1 element has been shown in several recent studies to serve as a binding site for the farnesoid X receptor (FXR), a nuclear receptor for bile acids. FXR activity requires heterodimerization with RXR alpha, and when bound by bile acids, the complex effectively regulates the transcription of several genes involved in bile acid homeostasis. Gel mobility shift assays demonstrated specific binding of FXR/RXR alpha heterodimers to the IR-1 element in the BSEP promoter. In HepG2 cells, co-transfection of FXR and RXR alpha is required to attain full transactivation of the BSEP promoter by bile acids. Two FXR transactivation-deficient mutants (an AF-2 deletion and a W469A point mutant) failed to transactivate, indicating that the effect of bile acids is FXR-dependent. Further, mutational analysis confirms that the FXR/RXR alpha heterodimer activates transcription through the IR-1 site in the human BSEP promoter. These results demonstrate a mechanism by which bile acids transcriptionally regulate the activity of the bile salt excretory pump, a critical component involved in the enterohepatic circulation of bile acids.

Reduction of atherosclerosis in apolipoprotein E knockout mice by activation of the retinoid X receptor.

A common feature of many metabolic pathways is their control by retinoid X receptor (RXR) heterodimers. Dysregulation of such metabolic pathways can lead to the development of atherosclerosis, a disease influenced by both systemic and local factors. Here we analyzed the effects of activation of RXR and some of its heterodimers in apolipoprotein E -/- mice, a well established animal model of atherosclerosis. An RXR agonist drastically reduced the development of atherosclerosis. In addition, a ligand for the peroxisome proliferator-activated receptor (PPAR)gamma and a dual agonist of both PPARalpha and PPARgamma had moderate inhibitory effects. Both RXR and liver X receptor (LXR) agonists induced ATP-binding cassette protein 1 (ABC-1) expression and stimulated ABC-1-mediated cholesterol efflux from macrophages from wild-type, but not from LXRalpha and beta double -/-, mice. Hence, activation of ABC-1-mediated cholesterol efflux by the RXR/LXR heterodimer might contribute to the beneficial effects of rexinoids on atherosclerosis and warrant further evaluation of RXR/LXR agonists in prevention and treatment of atherosclerosis.

LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes.

Apolipoprotein E (apoE) secreted by macrophages in the artery wall exerts an important protective effect against the development of atherosclerosis, presumably through its ability to promote lipid efflux. Previous studies have shown that increases in cellular free cholesterol levels stimulate apoE transcription in macrophages and adipocytes; however, the molecular basis for this regulation is unknown. Recently, Taylor and colleagues [Shih, S. J., Allan, C., Grehan, S., Tse, E., Moran, C. & Taylor, J. M. (2000) J. Biol. Chem. 275, 31567-31572] identified two enhancers from the human apoE gene, termed multienhancer 1 (ME.1) and multienhancer 2 (ME.2), that direct macrophage- and adipose-specific expression in transgenic mice. We demonstrate here that the nuclear receptors LXRalpha and LXRbeta and their oxysterol ligands are key regulators of apoE expression in both macrophages and adipose tissue. We show that LXR/RXR heterodimers regulate apoE transcription directly, through interaction with a conserved LXR response element present in both ME.1 and ME.2. Moreover, we demonstrate that the ability of oxysterols and synthetic ligands to regulate apoE expression in adipose tissue and peritoneal macrophages is reduced in Lxralpha-/- or Lxrbeta-/- mice and abolished in double knockouts. Basal expression of apoE is not compromised in Lxr null mice, however, indicating that LXRs mediate lipid-inducible rather than tissue-specific expression of this gene. Together with our previous work, these findings support a central role for LXR signaling pathways in the control of macrophage cholesterol efflux through the coordinate regulation of apoE, ABCA1, and ABCG1 expression.

Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta.

The liver X receptors (LXRs) are members of the nuclear hormone receptor superfamily that are bound and activated by oxysterols. These receptors serve as sterol sensors to regulate the transcription of gene products that control intracellular cholesterol homeostasis through catabolism and transport. In this report, we describe a novel LXR target, the sterol regulatory element-binding protein-1c gene (SREBP-1c), which encodes a membrane-bound transcription factor of the basic helix-loop-helix-leucine zipper family. SREBP-1c expression was markedly increased in mouse tissues in an LXR-dependent manner by dietary cholesterol and synthetic agonists for both LXR and its heterodimer partner, the retinoid X receptor (RXR). Expression of the related gene products, SREBP-1a and SREBP-2, were not increased. Analysis of the mouse SREBP-1c gene promoter revealed an RXR/LXR DNA-binding site that is essential for this regulation. The transcriptional increase in SREBP-1c mRNA by RXR/LXR was accompanied by a similar increase in the level of the nuclear, active form of the SREBP-1c protein and an increase in fatty acid synthesis. Because this active form of SREBP-1c controls the transcription of genes involved in fatty acid biosynthesis, our results reveal a unique regulatory interplay between cholesterol and fatty acid metabolism.

Role of LXRs in control of lipogenesis.

The discovery of oxysterols as the endogenous liver X receptor (LXR) ligands and subsequent gene targeting studies in mice provided strong evidence that LXR plays a central role in cholesterol metabolism. The identification here of a synthetic, nonsteroidal LXR-selective agonist series represented by T0314407 and T0901317 revealed a novel physiological role of LXR. Oral administration of T0901317 to mice and hamsters showed that LXR activated the coordinate expression of major fatty acid biosynthetic genes (lipogenesis) and increased plasma triglyceride and phospholipid levels in both species. Complementary studies in cell culture and animals suggested that the increase in plasma lipids occurs via LXR-mediated induction of the sterol regulatory element-binding protein 1 (SREBP-1) lipogenic program.

Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors.

The catabolism of cholesterol into bile acids is regulated by oxysterols and bile acids, which induce or repress transcription of the pathway's rate-limiting enzyme cholesterol 7alpha-hydroxylase (CYP7A1). The nuclear receptor LXRalpha binds oxysterols and mediates feed-forward induction. Here, we show that repression is coordinately regulated by a triumvirate of nuclear receptors, including the bile acid receptor, FXR; the promoter-specific activator, LRH-1; and the promoter-specific repressor, SHP. Feedback repression of CYP7A1 is accomplished by the binding of bile acids to FXR, which leads to transcription of SHP. Elevated SHP protein then inactivates LRH-1 by forming a heterodimeric complex that leads to promoter-specific repression of both CYP7A1 and SHP. These results reveal an elaborate autoregulatory cascade mediated by nuclear receptors for the maintenance of hepatic cholesterol catabolism.

The role of orphan nuclear receptors in the regulation of cholesterol homeostasis.

Cholesterol balance is maintained by a series of regulatory pathways that control the acquisition of cholesterol from endogenous and exogenous sources and the elimination of cholesterol, facilitated by its conversion to bile acids. Over the past decade, investigators have discovered that a family of membrane-bound transcription factors, sterol regulatory element-binding proteins (SREBPs), mediate the end-product repression of key enzymes of cholesterol biosynthesis. Recently orphan members of another family of transcription factors, the nuclear hormone receptors, have been found to regulate key pathways in bile acid metabolism, thereby controlling cholesterol elimination. The study of these orphan nuclear receptors suggests their potential as targets for new drug therapies.

A synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), is a ligand for the peroxisome proliferator-activated receptor gamma.

A novel synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), previously reported to have potent differentiating, antiproliferative, and antiinflammatory activities, has been identified as a ligand for the peroxisome proliferator-activated receptor gamma (PPARgamma). CDDO induces adipocytic differentiation in 3T3-L1 cells, although it is not as potent as the full agonist of PPARgamma, rosiglitazone. Binding studies of CDDO to PPARgamma using a scintillation proximity assay give a Ki between 10(-8) to 10(-7) M. In transactivation assays, CDDO is a partial agonist for PPARgamma. The methyl ester of CDDO, CDDO-Me, binds to PPARgamma with similar affinity, but is an antagonist. Like other PPARgamma ligands, CDDO synergizes with a retinoid X receptor (RXR)-specific ligand to induce 3T3-L1 differentiation, while CDDO-Me is an antagonist in this assay. The partial agonism of CDDO and the antagonism of CDDO-Me reflect the differences in their capacity to recruit or displace cofactors of transcriptional regulation; CDDO and rosiglitazone both release the nuclear receptor corepressor, NCoR, from PPARgamma, while CDDO-Me does not. The differences between CDDO and rosiglitazone as either partial or full agonists, respectively, are seen in the weaker ability of CDDO to recruit the coactivator CREB-binding protein, CBP, to PPARgamma. Our results establish the triterpenoid CDDO as a member of a new class of PPARgamma ligands.

Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers.

Several nuclear hormone receptors involved in lipid metabolism form obligate heterodimers with retinoid X receptors (RXRs) and are activated by RXR agonists such as rexinoids. Animals treated with rexinoids exhibited marked changes in cholesterol balance, including inhibition of cholesterol absorption and repressed bile acid synthesis. Studies with receptor-selective agonists revealed that oxysterol receptors (LXRs) and the bile acid receptor (FXR) are the RXR heterodimeric partners that mediate these effects by regulating expression of the reverse cholesterol transporter, ABC1, and the rate-limiting enzyme of bile acid synthesis, CYP7A1, respectively. Thus, these RXR heterodimers serve as key regulators of cholesterol homeostasis by governing reverse cholesterol transport from peripheral tissues, bile acid synthesis in liver, and cholesterol absorption in intestine.