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1.  Nuclear receptor LRH-1/NR5A2 is required and targetable for liver endoplasmic reticulum stress resolution 
eLife  null;3:e01694.
Chronic endoplasmic reticulum (ER) stress results in toxicity that contributes to multiple human disorders. We report a stress resolution pathway initiated by the nuclear receptor LRH-1 that is independent of known unfolded protein response (UPR) pathways. Like mice lacking primary UPR components, hepatic Lrh-1-null mice cannot resolve ER stress, despite a functional UPR. In response to ER stress, LRH-1 induces expression of the kinase Plk3, which phosphorylates and activates the transcription factor ATF2. Plk3-null mice also cannot resolve ER stress, and restoring Plk3 expression in Lrh-1-null cells rescues ER stress resolution. Reduced or heightened ATF2 activity also sensitizes or desensitizes cells to ER stress, respectively. LRH-1 agonist treatment increases ER stress resistance and decreases cell death. We conclude that LRH-1 initiates a novel pathway of ER stress resolution that is independent of the UPR, yet equivalently required. Targeting LRH-1 may be beneficial in human disorders associated with chronic ER stress.
eLife digest
A protein can only work properly if it has been folded into the correct shape. However, it is estimated that about one third of new proteins have the wrong shape. This is a major challenge for cells because misfolded proteins are often toxic, and cause many neurodegenerative and metabolic disorders.
In eukaryotic cells, most protein folding takes place inside a part of the cell called the endoplasmic reticulum (ER). If an incorrectly folded protein is detected, it is prevented from leaving the ER until it is refolded correctly, or destroyed. If too many proteins are misfolded, a process called the unfolded protein response helps the cell to cope with this ‘ER stress’ by expanding the ER and producing more of the molecules that assist protein folding. If this does not relieve the ER stress, the cell self-destructs. Neighboring cells then have to increase protein production to compensate for what would have been produced by the dead cell, thereby increasing the chance that they will also experience ER stress.
Activation of a protein called LRH-1 (short for liver receptor homolog-1) that is produced in the liver, pancreas and intestine can relieve the symptoms of the various metabolic diseases that are associated with chronic ER stress, including type II diabetes and fatty liver disease. However, researchers have been puzzled by the fact that although LRH-1 performs many different roles, its molecular structure provides few clues as to how it can do this.
Mamrosh et al. now confirm the speculated link between LRH-1 and ER stress relief in mice. LRH-1 triggers a previously unknown pathway that can relieve ER stress and is completely independent of the unfolded protein response. Targeting LRH-1 with certain chemical compounds alters its activity, suggesting that drug treatments could be developed to relieve ER stress. As similar targets for drugs have not been found in the unfolded protein response, the discovery of the LRH-1 pathway could lead to new approaches to the treatment of the diseases that result from ER stress.
PMCID: PMC3987120  PMID: 24737860
ER stress; nuclear receptors; liver metabolism; mouse
2.  MicroRNA-26a regulates insulin sensitivity and metabolism of glucose and lipids 
The Journal of Clinical Investigation  2015;125(6):2497-2509.
Type 2 diabetes (T2D) is characterized by insulin resistance and increased hepatic glucose production, yet the molecular mechanisms underlying these abnormalities are poorly understood. MicroRNAs (miRs) are a class of small, noncoding RNAs that have been implicated in the regulation of human diseases, including T2D. miR-26a is known to play a critical role in tumorigenesis; however, its function in cellular metabolism remains unknown. Here, we determined that miR-26a regulates insulin signaling and metabolism of glucose and lipids. Compared with lean individuals, overweight humans had decreased expression of miR-26a in the liver. Moreover, miR-26 was downregulated in 2 obese mouse models compared with control animals. Global or liver-specific overexpression of miR-26a in mice fed a high-fat diet improved insulin sensitivity, decreased hepatic glucose production, and decreased fatty acid synthesis, thereby preventing obesity-induced metabolic complications. Conversely, silencing of endogenous miR-26a in conventional diet–fed mice impaired insulin sensitivity, enhanced glucose production, and increased fatty acid synthesis. miR-26a targeted several key regulators of hepatic metabolism and insulin signaling. These findings reveal miR-26a as a regulator of liver metabolism and suggest miR-26a should be further explored as a potential target for the treatment of T2D.
PMCID: PMC4497741  PMID: 25961460
Endocrinology; Genetics; Metabolism
3.  Cysteine Sulfinic Acid Decarboxylase Regulation: A Role for FXR and SHP in Murine Hepatic Taurine Metabolism 
Bile acid synthesis is regulated by nuclear receptors including farnesoid X receptor (FXR) and small heterodimer partner (SHP), and by fibroblast growth factor15/19 (FGF15/19). Because bile acid synthesis involves amino acid conjugation, we hypothesized that hepatic cysteine sulfinic acid decarboxylase (CSAD) (a key enzyme in taurine synthesis) is regulated by bile acids.
To investigate CSAD regulation by bile acids and CSAD regulatory mechanisms.
Mice were fed a control diet or a diet supplemented with either 0.5% cholate or 2% cholestyramine. To gain mechanistic insight into CSAD regulation, we utilized GW4064 (FXR agonist), FGF19, or T-0901317 (LXR agonist) and Shp−/− mice. Tissue mRNA expression was determined by qRT-PCR. Amino acids were measured by HPLC.
Mice supplemented with dietary cholate exhibited reduced hepatic CSAD mRNA expression while those receiving cholestyramine exhibited increased hepatic CSAD mRNA expression. Activation of FXR suppressed CSAD mRNA expression whereas hepatic CSAD mRNA expression was increased in Shp−/− mice. Hepatic hypotaurine concentration (the product of CSAD) was higher in Shp−/− mice with a corresponding increase in serum (but not hepatic) taurine-conjugated bile acids. FGF19 administration suppressed hepatic CYP7A1 mRNA but did not change CSAD mRNA expression. LXR activation induced CYP7A1 mRNA yet failed to induce CSAD mRNA expression.
CSAD mRNA expression is physiologically regulated by bile acids in a feedback fashion via mechanisms involving SHP and FXR but not FGF15/19 or LXR. These novel findings implicate bile acids as regulators of CSAD mRNA via mechanisms shared in part with CYP7A1.
PMCID: PMC3995905  PMID: 24033844
Nuclear hormone receptors; taurine synthesis; bile acid metabolism; bile acid conjugation
5.  Nutrient Sensing Nuclear Receptors Coordinate Autophagy 
Nature  2014;516(7529):112-115.
Autophagy is an evolutionally conserved catabolic process that recycles nutrients upon starvation and maintains cellular energy homeostasis1–3. Its acute regulation by nutrient sensing signaling pathways is well described, but its longer-term transcriptional regulation is not. The nuclear receptors PPARα and FXR are activated in the fasted or fed liver, respectively4,5. Here we show that both regulate hepatic autophagy. Pharmacologic activation of PPARα reverses the normal suppression of autophagy in the fed state, inducing autophagic lipid degradation, or lipophagy. This response is lost in PPARα knockout (PPARα−/−) mice, which are partially defective in the induction of autophagy by fasting. Pharmacologic activation of the bile acid receptor FXR strongly suppresses the induction of autophagy in the fasting state, and this response is absent in FXR knockout (FXR−/−) mice, which show a partial defect in suppression of hepatic autophagy in the fed state. PPARα and FXR compete for binding to shared sites in autophagic gene promoters, with opposite transcriptional outputs. These results reveal complementary, interlocking mechanisms for regulation of autophagy by nutrient status.
PMCID: PMC4267857  PMID: 25383539
6.  Lysosomal Signaling Molecules Regulate Longevity in Caenorhabditis elegans 
Science (New York, N.Y.)  2015;347(6217):83-86.
Lysosomes are crucial cellular organelles for human health that function in digestion and recycling of extracellular and intracellular macromolecules. We describe a signaling role for lysosomes that affects aging. In the worm, Caenorhabditis elegans, the lysosomal acid lipase LIPL-4 triggered nuclear translocalization of a lysosomal lipid chaperone LBP-8, consequently promoting longevity by activating the nuclear hormone receptors NHR-49 and NHR-80. We used high-throughput metabolomic analysis to identify several lipids whose abundance was increased in worms constitutively over-expressing LIPL-4. Among them, oleoylethanolamide directly bound to LBP-8 and NHR-80 proteins, activated transcription of target genes of NHR-49 and NHR-80, and promoted longevity in C. elegans. These findings reveal a lysosome-to-nucleus signaling pathway that promotes longevity and suggest a function of lysosomes as signaling organelles in metazoans.
PMCID: PMC4425353  PMID: 25554789
7.  The orphan nuclear receptor small heterodimer partner is required for thiazolidinedione effects in leptin-deficient mice 
Small heterodimer partner (SHP, NR0B2) is involved in diverse metabolic pathways, including hepatic bile acid, lipid and glucose homeostasis, and has been implicated in effects on the peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and the receptor for antidiabetic drugs thiazolidinediones (TZDs). In this study, we aim to investigate the role of SHP in TZD response by comparing TZD-treated leptin-deficient (ob/ob) and leptin-, SHP-deficient (ob/ob;Shp−/−) double mutant mice.
Both ob/ob and double mutant ob/ob;Shp−/− mice developed hyperglycemia, insulin resistance, and hyperlipidemia, but hepatic fat accumulation was decreased in the double mutant ob/ob;Shp−/− mice. PPARγ2 mRNA levels were markedly lower in ob/ob;Shp−/− liver and decreased to a lesser extent in adipose tissue. The TZD troglitazone did not reduce glucose or circulating triglyceride levels in ob/ob;Shp−/− mice. Expression of the adipocytokines, such as adiponectin and resistin, was not stimulated by troglitazone treatment. Expression of hepatic lipogenic genes was also reduced in ob/ob;Shp−/− mice. Moreover, overexpression of SHP by adenovirus infection increased PPARγ2 mRNA levels in mouse primary hepatocytes.
Our results suggest that SHP is required for both antidiabetic and hypolipidemic effects of TZDs in ob/ob mice through regulation of PPARγ expression.
PMCID: PMC4489392  PMID: 25951943
SHP; Troglitazone; PPARγ; Thiazolidinedione
8.  All-trans-Retinoic Acid Ameliorates Hepatic Steatosis in Mice via a Novel Transcriptional Cascade 
Hepatology (Baltimore, Md.)  2014;59(5):1750-1760.
Mice deficient small heterodimer partner (SHP) are protected from diet induced hepatic steatosis due to increased fatty acid oxidation and decreased lipogenesis. The decreased lipogenesis appears to be a direct consequence of very low expression of peroxisome proliferator activated receptor gamma 2 (PPARγ2), a potent lipogenic transcription factor, in the SHP−/− liver. The current study focuses on the identification of a SHP dependent regulatory cascade that controls PPARγ2 gene expression, thereby regulating hepatic fat accumulation. Illumina BeadChip array and real-time polymerase chain reaction were used to identify genes responsible for the linkage between SHP and PPARγ2 using hepatic RNAs isolated from SHP−/− and SHP-overexpressing mice. The initial efforts identify that hairy and enhancer of split 6 (Hes6), a novel transcriptional repressor, is an important mediator of the regulation of PPARγ2 transcription by SHP. The Hes6 promoter is specifically activated by the retinoic acid receptor (RAR) in response to its natural agonist ligand all-trans retinoic acid (atRA), and is repressed by SHP. Hes6 subsequently represses hepatocyte nuclear factor 4 alpha (HNF4α) activated-PPARγ2 gene expression via direct inhibition of the HNF4α transcriptional activity. Furthermore, we provide evidences that atRA treatment or adenovirus-mediated RARα overexpression significantly reduced hepatic fat accumulation in obese mouse models as observed in earlier studies and the beneficial effect is achieved via the proposed transcriptional cascade.
Our study describes a novel transcriptional regulatory cascade controlling hepatic lipid metabolism that identifies retinoic acid signaling as a new therapeutic approach to non-alcoholic fatty liver diseases.
PMCID: PMC4008145  PMID: 24038081
Lipid homeostasis; Nuclear Hormone Receptors; Fsp27; SHP; Hes6
9.  Bile acids activate YAP to promote liver carcinogenesis 
Cell reports  2013;5(4):1060-1069.
Elevated bile acid levels increase hepatocellular carcinoma by unknown mechanisms. Here we show that mice with a severe defect in bile acid homeostasis due to loss of the nuclear receptors FXR and SHP have enlarged livers, progenitor cell proliferation, YAP (Yes Associated Protein) activation, and develop spontaneous liver tumorigenesis. This phenotype mirrors mice with loss of hippo kinases or overexpression of their downstream target YAP. Bile acids act as upstream regulators of YAP via a novel pathway dependent on induction of the scaffold protein Iqgap1. Patients with diverse biliary dysfunctions exhibit enhanced Iqgap1 and nuclear YAP expression. Our findings reveal an unexpected mechanism for bile acid regulation of liver growth and tumorigenesis via the Hippo pathway.
PMCID: PMC3961013  PMID: 24268772
10.  Opposing Regulation of Cytochrome P450 Expression by CAR and PXR in Hypothyroid Mice 
Toxicology and applied pharmacology  2012;263(2):131-137.
Clinical hypothyroidism affects various metabolic processes including drug metabolism. CYP2B and CYP3A are important cytochrome P450 drug metabolizing enzymes that are regulated by the xenobiotic receptors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2). We evaluated the regulation of the hepatic expression of CYPs by CAR and PXR in the hypothyroid state induced by a low-iodine diet containing 0.15% propylthiouracil. Expression of Cyp3a11 was suppressed in hypothyroid C57BL/6 wild type (WT) mice and a further decrement was observed in hypothyroid CAR-/- mice, but not in hypothyroid PXR-/- mice. In contrast, expression of Cyp2b10 was induced in both WT and PXR-/- hypothyroid mice, and this induction was abolished in CAR-/- mice and in and CAR-/- PXR-/- double knockouts. CAR mRNA expression was increased by hypothyroidism, while PXR expression remained unchanged. Carbamazepine (CBZ) is a commonly used antiepileptic that is metabolized by CYP3A isoforms. After CBZ treatment of normal chow fed mice, serum CBZ levels were highest in CAR-/- mice and lowest in WT and PXR-/- mice. Hypothyroid WT or PXR-/- mice survived chronic CBZ treatment, but all hypothyroid CAR-/- and CAR-/- PXR-/- mice died, with CAR-/-PXR-/- mice surviving longer than CAR-/- mice (12.3 ±3.3 days vs. 6.3 ±2.1 days, p=0.04). All these findings suggest that hypothyroid status affects xenobiotic metabolism, with opposing responses of CAR and PXR and their CYP targets that can cancel each other out, decreasing serious metabolic derangement in response to a xenobiotic challenge.
PMCID: PMC3399920  PMID: 22503787
thyroid hormone; cytochrome P450; xenobiotic receptor
11.  A Common Thread Connects Antidiabetic Effects of Multiple Nuclear Receptors 
Cell metabolism  2012;15(5):615-622.
Several pathways and pathologies have been suggested as connections between obesity and diabetes, including inflammation of adipose and other tissues, toxic lipids, endoplasmic reticulum stress, and fatty liver. One specific proposal is that insulin resistance induces a vicious cycle in which hyperinsulinemia increases hepatic lipogenesis and exacerbates fatty liver, in turn further increasing insulin resistance. Here I suggest that reversing this cycle via suppression of the lipogenic transcription factor SREBP-1c is a common thread that connects the antidiabetic effects of a surprising number of nuclear hormone receptors, including CAR, LRH-1, TRβ, ERα and FXR/SHP.
PMCID: PMC3613429  PMID: 22560214
Consumption of dietary flavonoids has been associated with reduced mortality and risk of cardiovascular disease, partially by reducing triglyceridemia. We have previously reported that a grape seed procyanidin extract (GSPE) reduces postprandial triglyceridemia in normolipidemic animals signaling through the orphan nuclear receptor Small Heterodimer Partner (SHP) a target of the bile acid receptor Farnesoid X Receptor (FXR). Our aim was to elucidate whether FXR mediates the hypotriglyceridemic effect of procyanidins. In FXR-driven luciferase expression assays GSPE dose-dependently enhanced FXR activity in the presence of chenodeoxycholic acid. GSPE gavage reduced triglyceridemia in wild type mice but not in FXR-null mice, revealing FXR as an essential mediator of the hypotriglyceridemic actions of procyanidins in vivo. In the liver, GSPE down-regulated, in a FXR-dependent manner, the expression of the transcription factor Steroid Response Element Binding Protein 1 (SREBP1) and several SREBP1 target genes involved in lipogenesis, and upregulated ApoA5 expression. Altogether, our results indicate that procyanidins lower triglyceridemia following the same pathway as bile acids: activation of FXR, transient upregulation of SHP expression and subsequent downregulation of SREBP1 expression. This study adds dietary procyanidins to the arsenal of FXR ligands with potential therapeutic use to combat hypertriglyceridemia, type 2 diabetes and metabolic syndrome.
PMCID: PMC4142053  PMID: 19496086
Bile Acids; FXR; liver; procyanidins; SHP; SBARM; SREBP1; triglycerides
13.  Constitutive Androstane Receptor Activation Decreases Plasma Apolipoprotein B–Containing Lipoproteins and Atherosclerosis in Low-Density Lipoprotein Receptor–Deficient Mice 
The goal of this study was to determine the impact of the nuclear receptor constitutive androstane receptor (CAR) on lipoprotein metabolism and atherosclerosis in hyperlipidemic mice.
Methods and Results
Low-density lipoprotein receptor–deficient (Ldlr−/−) and apolipoprotein E–deficient (ApoE−/−) mice fed a Western-type diet were treated weekly with the Car agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or the vehicle only for 8 weeks. In Ldlr−/− mice, treatment with TCPOBOP induced a decrease in plasma triglyceride and intermediate-density lipoprotein/low-density lipoprotein cholesterol levels (≈30% decrease in both cases after 2 months, P<0.01). These mice also showed a significant reduction in the production of very-low-density lipoproteins associated with a decrease in hepatic triglyceride content and the repression of several genes involved in lipogenesis. TCPOBOP treatment also induced a marked increase in the very-low-density lipoprotein receptor in the liver, which probably contributed to the decrease in intermediate-density lipoprotein/low-density lipoprotein levels. Atherosclerotic lesions in the aortic valves of TCPOBOP-treated Ldlr−/− mice were also reduced (−60%, P<0.001). In ApoE−/− mice, which lack the physiological apoE ligand for the very-low-density lipoprotein receptor, the effect of TCPOBOP on plasma cholesterol levels and the development of atherosclerotic lesions was markedly attenuated.
CAR is a potential target in the prevention and treatment of hypercholesterolemia and atherosclerosis.
PMCID: PMC4104927  PMID: 21778422
atherosclerosis; lipoproteins; constitutive androstane receptor
14.  FXR-induced secretion of FGF15/19 inhibits CYP27 expression in cholangiocytes through p38 kinase pathway 
Cholangiocytes, bile duct lining cells, actively adjust the amount of cholesterol and bile acids in bile through expression of enzymes and channels involved in transportation and metabolism of the cholesterol and bile acids. Herein, we report molecular mechanisms regulating bile acid biosynthesis in cholangiocytes. Among the cytochrome p450 (Cyp) enzymes involved in bile acid biosynthesis, sterol 27-hydroxylase (Cyp27) that is the rate-limiting enzyme for the acidic pathway of bile acid biosynthesis expressed in cholangiocytes. Expression of other Cyp enzymes for the basic bile acid biosynthesis was hardly detected. The Cyp27 expression was negatively regulated by a hydrophobic bile acid through farnesoid X receptor (FXR), a nuclear receptor activated by bile acid ligands. Activated FXR exerted the negative effects by inducing an expression of fibroblast growth factor 15/19 (FGF15/19). Similar to its repressive function against cholesterol 7α-hydroxylase (Cyp7a1) expression in hepatocytes, secreted FGF15/19 triggered Cyp27 repression in cholangiocytes through interaction with its cognate receptor fibroblast growth factor receptor 4 (FGFR4). The involvements of FXR and FGFR4 for the bile acid-induced Cyp27 repression were confirmed in vivo using knockout mouse models. Different from the signaling in hepatocytes, wherein the FGF15/19-induced repression signaling is mediated by c-Jun N-terminal kinase (JNK), FGF15/19-induced Cyp27 repression in cholangiocytes was mediated by p38 kinase. Thus, the results collectively suggest that cholangiocytes may be able to actively regulate bile acid biosynthesis in cholangiocytes and even hepatocyte by secreting FGF15/19. We suggest the presence of cholangiocyte-mediated intrahepatic feedback loop in addition to the enterohepatic feedback loop against bile acid biosynthesis in the liver.
PMCID: PMC3967003  PMID: 24068255
Cholangiocyte; Cyp27; p38 kinase
15.  Combined deletion of Fxr and Shp in mice induces Cyp17a1 and results in juvenile onset cholestasis  
Bile acid homeostasis is tightly regulated via a feedback loop operated by the nuclear receptors farnesoid X receptor (FXR) and small heterodimer partner (SHP). Contrary to current models, which place FXR upstream of SHP in a linear regulatory pathway, here we show that the phenotypic consequences in mice of the combined loss of both receptors are much more severe than the relatively modest impact of the loss of either Fxr or Shp alone. Fxr–/–Shp–/– mice exhibited cholestasis and liver injury as early as 3 weeks of age, and this was linked to the dysregulation of bile acid homeostatic genes, particularly cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1). In addition, double-knockout mice showed misregulation of genes in the C21 steroid biosynthesis pathway, with strong induction of cytochrome P450, family 17, subfamily a, polypeptide 1 (Cyp17a1), resulting in elevated serum levels of its enzymatic product 17-hydroxyprogesterone (17-OHP). Treatment of WT mice with 17-OHP was sufficient to induce liver injury that reproduced many of the histopathological features observed in the double-knockout mice. Therefore, our data indicate a pathologic role for increased production of 17-hydroxy steroid metabolites in liver injury and suggest that Fxr–/–Shp–/– mice could provide a model for juvenile onset cholestasis.
PMCID: PMC3007143  PMID: 21123943
16.  Liver X Receptor β and Peroxisome Proliferator-Activated Receptor δ regulate cholesterol transport in cholangiocytes 
Hepatology (Baltimore, Md.)  2012;56(6):2288-2296.
Nuclear receptors (NRs) play crucial roles in regulation of hepatic cholesterol synthesis, metabolism and conversion to bile acids, but their actions in cholangiocytes have not been examined. In this study, we investigated the roles of NRs in cholangiocyte physiology and cholesterol metabolism and flux. We examined the expression of NRs and other genes involved in cholesterol homeostasis in freshly isolated and cultured rodent cholangiocytes and found that these cells express a specific subset of NRs which includes Liver X Receptor β (LXRβ) and Peroxisome Proliferator-Activated Receptor δ (PPARδ). Activation of LXRβ and/or PPARδ in cholangiocytes induces ATP-binding cassette cholesterol transporter A1 (ABCA1) and increases cholesterol export at the basolateral compartment in polarized cultured cholangiocytes. In addition, PPARδ induces Niemann Pick C1 Like L1 (NPC1L1), which imports cholesterol into cholangiocytes and is expressed on the apical cholangiocyte membrane, via specific interaction with a PPRE within the NPC1L1 promoter. Based on these studies, we propose that (i) LXRβ and PPARδ coordinate NPC1L1/ABCA1 dependent vectorial cholesterol flux from bile through cholangiocytes and (ii) manipulation of these processes may influence bile composition with important applications in cholestatic liver disease and gallstone disease, serious health concerns for humans.
PMCID: PMC3469731  PMID: 22729460
Cholangiocyte; LXRβ; PPARδ; ABCA1; NPC1L1
17.  Bmal1 and β-Cell Clock Are Required for Adaptation to Circadian Disruption, and Their Loss of Function Leads to Oxidative Stress-Induced β-Cell Failure in Mice 
Molecular and Cellular Biology  2013;33(11):2327-2338.
Circadian disruption has deleterious effects on metabolism. Global deletion of Bmal1, a core clock gene, results in β-cell dysfunction and diabetes. However, it is unknown if this is due to loss of cell-autonomous function of Bmal1 in β cells. To address this, we generated mice with β-cell clock disruption by deleting Bmal1 in β cells (β-Bmal1−/−). β-Bmal1−/− mice develop diabetes due to loss of glucose-stimulated insulin secretion (GSIS). This loss of GSIS is due to the accumulation of reactive oxygen species (ROS) and consequent mitochondrial uncoupling, as it is fully rescued by scavenging of the ROS or by inhibition of uncoupling protein 2. The expression of the master antioxidant regulatory factor Nrf2 (nuclear factor erythroid 2-related factor 2) and its targets, Sesn2, Prdx3, Gclc, and Gclm, was decreased in β-Bmal1−/− islets, which may contribute to the observed increase in ROS accumulation. In addition, by chromatin immunoprecipitation experiments, we show that Nrf2 is a direct transcriptional target of Bmal1. Interestingly, simulation of shift work-induced circadian misalignment in mice recapitulates many of the defects seen in Bmal1-deficient islets. Thus, the cell-autonomous function of Bmal1 is required for normal β-cell function by mitigating oxidative stress and serves to preserve β-cell function in the face of circadian misalignment.
PMCID: PMC3648066  PMID: 23547261
18.  Orphan Receptor Small Heterodimer Partner Suppresses Tumorigenesis by Modulating Cyclin D1 Expression and Cellular Proliferation 
Hepatology (Baltimore, Md.)  2008;48(1):289-298.
The small heterodimer partner (SHP; NROB2), a member of the nuclear receptor superfamily, contributes to the biological regulation of several major functions of the liver. However, the role of SHP in cellular proliferation and tumorigenesis has not been investigated before. Here we report that SHP negatively regulates tumorigenesis both in vivo and in vitro. SHP−/− mice aged 12 to 15 months old developed spontaneous hepatocellular carcinoma, which was found to be strongly associated with enhanced hepatocyte proliferation and increased cyclin D1 expression. In contrast, overexpressing SHP in hepatocytes of SHP-transgenic mice reversed this effect. Embryonic fibroblasts lacking SHP showed enhanced proliferation and produced increased cyclin D1 messenger RNA and protein, and SHP was shown to be a direct negative regulator of cyclin D1 gene transcription. The immortal SHP−/− fibroblasts displayed characteristics of malignant transformed cells and formed tumors in nude mice.
These results provide first evidence that SHP plays tumor suppressor function by negatively regulating cellular growth.
PMCID: PMC3800167  PMID: 18537191
19.  Neonatal activation of the nuclear receptor CAR results in epigenetic memory and permanent change of drug metabolism in mouse liver 
Hepatology (Baltimore, Md.)  2012;56(4):1499-1508.
Aberrant epigenetic alterations during development may result in long-term epigenetic memory and have a permanent effect on the health of subjects. Constitutive androstane receptor (CAR; NR1I3) is a central regulator of drug/xenobiotic metabolism. Here, we report that transient neonatal activation of CAR results in epigenetic memory and a permanent change of liver drug metabolism. CAR activation by neonatal exposure to a CAR-specific ligand, 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) led to persistently induced expression of the CAR target genes Cyp2B10 and Cyp2C37 throughout the life of exposed mice. These mice showed a permanent reduction in sensitivity to zoxazolamine treatment as adults. Compared with control groups, the induction of Cyp2B10 and Cyp2C37 in hepatocytes isolated from these mice was more sensitive to low concentrations of the CAR agonist TCPOBOP. Accordingly, neonatal activation of CAR led to a permanent increase of histone 3 lysine 4 (H3K4) mono-, di- and trimethylation and decrease of H3K9 trimethylation within the Cyp2B10 locus. Transcriptional coactivator ASC-2 and histone demethylase JMJD2d participated in this CAR-dependent epigenetic switch.
Neonatal activation of CAR results in epigenetic memory and a permanent change of liver drug metabolism.
PMCID: PMC3407349  PMID: 22488010
CAR; nuclear receptor; epigenetic memory; drug metabolism; histone methylation
20.  The Nrf2 Activator Oltipraz Also Activates the Constitutive Androstane Receptor 
Oltipraz (OPZ) is a well known inducer of NAD(P)H:quinone oxidoreductase (NQO1) along with other enzymes that comprise the nuclear factor E2-related factor 2 (Nrf2) battery of detoxification genes. However, OPZ treatment also induces expression of CYP2B, a gene regulated by the constitutive androstane receptor (CAR). Therefore, this study was designed to determine whether OPZ induces gene expression in the mouse liver through activation of CAR in addition to Nrf2. OPZ increased the mRNA expression of both Cyp2b10 and Nqo1 in C57BL/6 mouse livers. As expected, in livers from Nrf2−/− mice, OPZ induction of Nqo1 was reduced, indicating Nqo1 induction is dependent on Nrf2 activation, whereas Cyp2b10 induction was unchanged. The robust induction of Cyp2b10 by OPZ in wild-type mice was completely absent in CAR−/− mice, revealing a CAR-dependent induction by OPZ. OPZ also induced transcription of the human CYP2B6 promoter-reporter containing the phenobarbital (PB) responsive element in mouse liver using an in vivo transcription assay. Additionally, OPZ induced in vivo nuclear accumulation of CAR at 3 h but, as with PB, was unable to reverse androstanol repression of mouse CAR constitutive activity in transiently transfected HepG2 cells. In summary, OPZ induces expression of Cyp2b10 and Nqo1 via the activation of CAR and Nrf2, respectively.
PMCID: PMC3693743  PMID: 18474683
21.  Farnesoid X receptor is essential for normal glucose homeostasis 
Journal of Clinical Investigation  2006;116(4):1102-1109.
The bile acid receptor farnesoid X receptor (FXR; NR1H4) is a central regulator of bile acid and lipid metabolism. We show here that FXR plays a key regulatory role in glucose homeostasis. FXR-null mice developed severe fatty liver and elevated circulating FFAs, which was associated with elevated serum glucose and impaired glucose and insulin tolerance. Their insulin resistance was confirmed by the hyperinsulinemic euglycemic clamp, which showed attenuated inhibition of hepatic glucose production by insulin and reduced peripheral glucose disposal. In FXR–/– skeletal muscle and liver, multiple steps in the insulin signaling pathway were markedly blunted. In skeletal muscle, which does not express FXR, triglyceride and FFA levels were increased, and we propose that their inhibitory effects account for insulin resistance in that tissue. In contrast to the results in FXR–/– mice, bile acid activation of FXR in WT mice repressed expression of gluconeogenic genes and decreased serum glucose. The absence of this repression in both FXR–/– and small heterodimer partner–null (SHP–/–) mice demonstrated that the previously described FXR-SHP nuclear receptor cascade also targets glucose metabolism. Taken together, our results identify a link between lipid and glucose metabolism mediated by the FXR-SHP cascade.
PMCID: PMC1409738  PMID: 16557297
22.  Loss of Bmal1 leads to uncoupling and impaired glucose-stimulated insulin secretion in β-cells 
Islets  2011;3(6):381-388.
The circadian clock has been shown to regulate metabolic homeostasis. Mice with a deletion of Bmal1, a key component of the core molecular clock, develop hyperglycemia and hypoinsulinemia suggesting β-cell dysfunction. However, the underlying mechanisms are not fully known. In this study, we investigated the mechanisms underlying the regulation of β-cell function by Bmal1. We studied β-cell function in global Bmal1-/- mice, in vivo and in isolated islets ex vivo, as well as in rat insulinoma cell lines with shRNA-mediated Bmal1 knockdown. Global Bmal1-/- mice develop diabetes secondary to a significant impairment in glucose-stimulated insulin secretion (GSIS). There is a blunting of GSIS in both isolated Bmal1-/- islets and in Bmal1 knockdown cells, as compared with controls, suggesting that this is secondary to a loss of cell-autonomous effect of Bmal1. In contrast to previous studies, in these Bmal1-/- mice on a C57Bl/6 background, the loss of stimulated insulin secretion, interestingly, is with glucose but not to other depolarizing secretagogues, suggesting that events downstream of membrane depolarization are largely normal in Bmal1-/- islets. This defect in GSIS occurs as a result of increased mitochondrial uncoupling with consequent impairment of glucose-induced mitochondrial potential generation and ATP synthesis, due to an upregulation of Ucp2. Inhibition of Ucp2 in isolated islets leads to a rescue of the glucose-induced ATP production and insulin secretion in Bmal1-/- islets. Thus, Bmal1 regulates mitochondrial energy metabolism to maintain normal GSIS and its disruption leads to diabetes due to a loss of GSIS.
PMCID: PMC3329519  PMID: 22045262
Bmal1; circadian clock; diabetes; insulin secretion; mitochondria; β-cells
23.  Limited Effects of Bile Acids and Small Heterodimer Partner on Hepatitis B Virus Biosynthesis In Vivo 
Journal of Virology  2012;86(5):2760-2768.
Multiple nuclear receptors, including hepatocyte nuclear factor 4α (HNF4α), retinoid X receptor α (RXRα) plus peroxisome proliferator-activated receptor α (PPARα), RXRα plus farnesoid X receptor α (FXRα), liver receptor homolog 1 (LRH1), and estrogen-related receptors (ERRs), have been shown to support efficient viral biosynthesis in nonhepatoma cells in the absence of additional liver-enriched transcription factors. Although HNF4α has been shown to be critical for the developmental expression of hepatitis B virus (HBV) biosynthesis in the liver, the relative importance of the various nuclear receptors capable of supporting viral transcription and replication in the adult in vivo has not been clearly established. To investigate the role of the nuclear receptor FXR and the corepressor small heterodimer partner (SHP) in viral biosynthesis in vivo, SHP-expressing and SHP-null HBV transgenic mice were fed a bile acid-supplemented diet. The increased FXR activity and SHP expression levels resulting from bile acid treatment did not greatly modulate HBV RNA and DNA synthesis. Therefore, FXR and SHP appear to play a limited role in modulating HBV biosynthesis, suggesting that alternative nuclear receptors are more critical determinants of viral transcription in the HBV transgenic mouse model of chronic viral infection. These observations suggest that hepatic bile acid levels or therapeutic agents targeting FXR may not greatly modulate viremia during natural infection.
PMCID: PMC3302260  PMID: 22171277
24.  Ortho-Aminoazotoluene activates mouse Constitutive Androstane Receptor (mCAR) and increases expression of mCAR target genes 
2'-3-dimethyl-4-aminoazobenzene (ortho-aminoazotoluene, OAT) is an azo dye and a rodent carcinogen that has been evaluated by the International Agency for Research on Cancer (IARC) as a possible (class 2B) human carcinogen. Its mechanism of action remains unclear. We examined the role of the xenobiotic receptor Constitutive Androstane Receptor (CAR, NR1I3) as a mediator of the effects of OAT. We found that OAT increases mouse CAR (mCAR) transactivation in a dose-dependent manner. This effect is specific because another closely related azo dye, 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB), did not activate mCAR. Real-time Q-PCR analysis in wild-type C57BL/6 mice revealed that OAT induces the hepatic mRNA expression of the following CAR target genes: Cyp2b10, Cyp2c29, Cyp3a11, Ugt1a1, Mrp4, Mrp2 and c-Myc. CAR-null (Car−/−) mice showed no increased expression of these genes following OAT treatment, demonstrating that CAR is required for their OAT dependent induction. The OAT-induced CAR-dependent increase of Cyp2b10 and c-Myc expression was confirmed by Western blotting. Immunohistochemistry analysis of wild-type and Car−/− livers showed that OAT did not acutely induce hepatocyte proliferation, but at much later time points showed an unexpected CAR-dependent proliferative response. These studies demonstrate that mCAR is an OAT xenosensor, and indicate that at least some of the biological effects of this compound are mediated by this nuclear receptor.
PMCID: PMC3148291  PMID: 21672546
Ortho-Aminoazotoluene (OAT); Constitutive Androstane Receptor (CAR); CYP450s; c-Myc; hepatocyte proliferation
25.  A traditional herbal medicine enhances bilirubin clearance by activating the nuclear receptor CAR 
Yin Zhi Huang, a decoction of Yin Chin (Artemisia capillaris) and three other herbs, is widely used in Asia to prevent and treat neonatal jaundice. We recently identified the constitutive androstane receptor (CAR, NR1I3) as a key regulator of bilirubin clearance in the liver. Here we show that treatment of WT and humanized CAR transgenic mice with Yin Zhi Huang for 3 days accelerates the clearance of intravenously infused bilirubin. This effect is absent in CAR knockout animals. Expression of bilirubin glucuronyl transferase and other components of the bilirubin metabolism pathway is induced by Yin Zhi Huang treatment of WT mice or mice expressing only human CAR, but not CAR knockout animals. 6,7-Dimethylesculetin, a compound present in Yin Chin, activates CAR in primary hepatocytes from both WT and humanized CAR mice and accelerates bilirubin clearance in vivo. We conclude that CAR mediates the effects of Yin Zhi Huang on bilirubin clearance and that 6,7-dimethylesculetin is an active component of this herbal medicine. CAR is a potential target for the development of new drugs to treat neonatal, genetic, or acquired forms of jaundice.
PMCID: PMC300765  PMID: 14702117

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