LXRα Regulates ChREBPα Transactivity in a Target Gene-Specific Manner through an Agonist-Modulated LBD-LID Interaction.

The cholesterol-sensing nuclear receptor liver X receptor (LXR) and the glucose-sensing transcription factor carbohydrate responsive element-binding protein (ChREBP) are central players in regulating glucose and lipid metabolism in the liver. More knowledge of their mechanistic interplay is needed to understand their role in pathological conditions like fatty liver disease and insulin resistance. In the current study, LXR and ChREBP co-occupancy was examined by analyzing ChIP-seq datasets from mice livers. LXR and ChREBP interaction was determined by Co-immunoprecipitation (CoIP) and their transactivity was assessed by real-time quantitative polymerase chain reaction (qPCR) of target genes and gene reporter assays. Chromatin binding capacity was determined by ChIP-qPCR assays. Our data show that LXRα and ChREBPα interact physically and show a high co-occupancy at regulatory regions in the mouse genome. LXRα co-activates ChREBPα and regulates ChREBP-specific target genes in vitro and in vivo. This co-activation is dependent on functional recognition elements for ChREBP but not for LXR, indicating that ChREBPα recruits LXRα to chromatin in trans. The two factors interact via their key activation domains; the low glucose inhibitory domain (LID) of ChREBPα and the ligand-binding domain (LBD) of LXRα. While unliganded LXRα co-activates ChREBPα, ligand-bound LXRα surprisingly represses ChREBPα activity on ChREBP-specific target genes. Mechanistically, this is due to a destabilized LXRα:ChREBPα interaction, leading to reduced ChREBP-binding to chromatin and restricted activation of glycolytic and lipogenic target genes. This ligand-driven molecular switch highlights an unappreciated role of LXRα in responding to nutritional cues that was overlooked due to LXR lipogenesis-promoting function.

Characterization of TCDD-inducible poly-ADP-ribose polymerase (TIPARP/ARTD14) catalytic activity.

Here, we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo-p-dioxin poly-ADP-ribose polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400-657 as the minimum catalytically active region, which retained its ability to mono-ADP-ribosylate AHR. However, the ability of TIPARP to ADP-ribosylate and repress AHR in cells was dependent on both its catalytic activity and zinc finger domain. The catalytic activity of TIPARP was resistant to meta-iodobenzylguanidine but sensitive to iodoacetamide and hydroxylamine, implicating cysteines and acidic side chains as ADP-ribosylated target residues. Mass spectrometry identified multiple ADP-ribosylated peptides in TIPARP and AHR. Electron transfer dissociation analysis of the TIPARP peptide 33ITPLKTCFK41 revealed cysteine 39 as a site for mono-ADP-ribosylation. Mutation of cysteine 39 to alanine resulted in a small, but significant, reduction in TIPARP autoribosylation activity, suggesting that additional amino acid residues are modified, but loss of cysteine 39 did not prevent its ability to repress AHR. Our findings characterize the subcellular localization and mono-ADP-ribosyltransferase activity of TIPARP, identify cysteine as a mono-ADP-ribosylated residue targeted by this enzyme, and confirm the TIPARP-dependent mono-ADP-ribosylation of other protein targets, such as AHR.

TCDD-inducible poly-ADP-ribose polymerase (TIPARP/PARP7) mono-ADP-ribosylates and co-activates liver X receptors.

Members of the poly-ADP-ribose polymerase (PARP) family catalyse the ADP-ribosylation of target proteins and are known to play important roles in many cellular processes, including DNA repair, differentiation and transcription. The majority of PARPs exhibit mono-ADP-ribosyltransferase activity rather than PARP activity; however, little is known about their biological activity. In the present study, we report that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-ADP-ribose polymerase (TIPARP), mono-ADP-ribosylates and positively regulates liver X receptor α (LXRα) and LXRß activity. Overexpression of TIPARP enhanced LXR-reporter gene activity. TIPARP knockdown or deletion reduced LXR regulated target gene expression levels in HepG2 cells and in Tiparp(-/-)mouse embryonic fibroblasts (MEFs) respectively. Deletion and mutagenesis studies showed that TIPARP's zinc-finger and catalytic domains were required to enhance LXR activity. Protein interaction studies using TIPARP and LXRα/ß peptide arrays revealed that LXRs interacted with an N-terminal sequence (a.a. 209-236) of TIPARP, which also overlapped with a putative co-activator domain of TIPARP (a.a. 200-225). Immunofluorescence studies showed that TIPARP and LXRα or LXRß co-localized in the nucleus.In vitroribosylation assays provided evidence that TIPARP mono-ADP-ribosylated both LXRα and LXRß. Co-immunoprecipitation (co-IP) studies revealed that ADP-ribosylase macrodomain 1 (MACROD1), but not MACROD2, interacted with LXRs in a TIPARP-dependent manner. This was complemented by reporter gene studies showing that MACROD1, but not MACROD2, prevented the TIPARP-dependent increase in LXR activity. GW3965-dependent increases in hepatic Srebp1 mRNA and protein expression levels were reduced in Tiparp(-/-)mice compared with Tiparp(+/+)mice. Taken together, these data identify a new mechanism of LXR regulation that involves TIPARP, ADP-ribosylation and MACROD1.

Activation of Liver X receptors in the heart leads to accumulation of intracellular lipids and attenuation of ischemia-reperfusion injury.

Liver X receptor (LXR)-α and -ß play a major role in lipid and glucose homeostasis. Their expression and function in the heart is not well characterized. Our aim was to describe the expression of LXRs in the murine heart, and to determine effects of cardiac LXR activation on target gene expression, lipid homeostasis and ischemia. Both LXRα and -ß were expressed in heart tissues, HL-1 cells and isolated cardiomyocytes as determined by qRT-PCR. Elevated cardiac expression of LXR target genes and LXRß was observed 24 h after in vivo permanent coronary artery ligation. The synthetic LXR agonist GW3965 induced mRNA expression of the LXR target genes in HL-1 cells and isolated cardiomyocytes. This was associated with a buildup of intracellular triglycerides and expanding lipid droplets as quantified by confocal microscopy. Mice injected with GW3965 had cardiac LXR activation as judged by increased target gene expression and lipid droplet accumulation. GW3965 in vivo and in vitro increased expression of genes inducing triglyceride synthesis, and altered expression of lipid droplet-binding protein genes. GW3965 protected HL-1 cells against hypoxia-reoxygenation induced apoptosis. LXR activation by GW3965 in vivo prior to heart isolation and perfusion with induced global ischemia and reperfusion improved left ventricular contractile function and decreased infarct size. In conclusion, LXRs are expressed in the murine heart in the basal state, and are activated by myocardial infarction. Activation of LXR by the synthetic agonist GW3965 is associated with intracardiac accumulation of lipid droplets and protection against myocardial ischemia-reperfusion injury.

Activation of LXR increases acyl-CoA synthetase activity through direct regulation of ACSL3 in human placental trophoblast cells.

Placental fatty acid transport and metabolism are important for proper growth and development of the feto-placental unit. The nuclear receptors, liver X receptors alpha and beta (LXRalpha and LXRbeta), are key regulators of lipid metabolism in many tissues, but little is known about their role in fatty acid transport and metabolism in placenta. The current study investigates the LXR-mediated regulation of long-chain acyl-CoA synthetase 3 (ACSL3) and its functions in human placental trophoblast cells. We demonstrate that activation of LXR increases ACSL3 expression, acyl-CoA synthetase activity, and fatty acid uptake in human tropholast cells. Silencing of ACSL3 in these cells attenuates the LXR-mediated increase in acyl-CoA synthetase activity. Furthermore, we show that ACSL3 is directly regulated by LXR through a conserved LXR responsive element in the ACSL3 promoter. Our results suggest that LXR plays a regulatory role in fatty acid metabolism by direct regulation of ACSL3 in human placental trophoblast cells.

Regulation of ADRP expression by long-chain polyunsaturated fatty acids in BeWo cells, a human placental choriocarcinoma cell line.

Transplacental transfer of maternal fatty acids is critical for fetal growth and development. In the placenta, a preferential uptake of fatty acids toward long-chain polyunsaturated fatty acids (LCPUFAs) has been demonstrated. Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that has been ascribed a role in cellular fatty acid uptake and storage. However, its role in placenta is not known. We demonstrate that ADRP mRNA and protein are regulated by fatty acids in a human placental choriocarcinoma cell line (BeWo) and in primary human trophoblasts. LCPUFAs of the n-3 and n-6 series [arachidonic acid (20:4n-6), docosahexaenoic acid (22:6n-3), and eicosapentaenoic acid (20:5n-3)] were more efficient than shorter fatty acids at stimulating ADRP mRNA expression. The fatty acid-mediated increase in ADRP mRNA expression was not related to the differentiation state of the cells. Synthetic peroxisome proliferator-activated receptor and retinoic X receptor agonists increased ADRP mRNA level but had no effect on ADRP protein level in undifferentiated BeWo cells. Furthermore, we show that incubation of BeWo cells with LCPUFAs, but not synthetic agonists, increased the cellular content of radiolabeled oleic acid, coinciding with the increase in ADRP mRNA and protein level. These studies provide new information on the regulation of ADRP in placental trophoblasts and suggest that LCPUFA-dependent regulation of ADRP could be involved in the metabolism of lipids in the placenta.

LXR is crucial in lipid metabolism.

Liver X receptors (LXRalpha and LXRbeta) are members of the nuclear receptor superfamily and are activated by oxysterols and intermediates in the cholesterol synthetic pathway. The pivotal role of LXRs in the metabolic conversion of cholesterol to bile acids is well established. Analysis of gene expression in LXRalpha and LXRbeta deficient mice have confirmed that LXR regulates a number of target genes involved in both cholesterol and fatty acid metabolism in liver, macrophages and intestine. The observation that LXRalpha is responsive to fatty acids and is expressed in metabolic tissues suggests that it also plays a general role in lipid metabolism. Adipose tissue is the main storage site for fat in the body and plays a crucial role in overall lipid handling. Both LXRalpha and LXRbeta are expressed and activated by endogenous and synthetic ligands, which lead to lipid accumulation into adipocytes. This indicates an important regulatory role of LXR in several metabolic signaling pathways in the adipose tissue, such as glucose uptake and de novo fatty acid synthesis. Here, we review recent studies that provide new insights into the mechanisms by which LXRs act to influence fatty acid synthesis in liver and adipose tissue.

Sulfur-substituted and alpha-methylated fatty acids as peroxisome proliferator-activated receptor activators.

FA with varying chain lengths and an alpha-methyl group and/or a sulfur in the beta-position were tested as peroxisome proliferator-activated receptor (PPAR)alpha, -delta(beta), and -gamma ligands by transient transfection in COS-1 cells using chimeric receptor expression plasmids, containing cDNAs encoding the ligand-binding domain of PPARalpha, -delta, and -gamma. For PPARalpha, an increasing activation was found with increasing chain length of the sulfur-substituted FA up to C14-S acetic acid (tetradecylthioacetic acid = TTA). The derivatives were poor, and nonsignificant, activators of PPARdelta. For PPARgamma, activation increased with increasing chain length up to C16-S acetic acid. A methyl group was introduced in the alpha-position of palmitic acid, TTA, EPA, DHA, cis9,trans11 CLA, and trans10,cis12 CLA. An increased activation of PPARalpha was obtained for the alpha-methyl derivatives compared with the unmethylated FA. This increase also resulted in increased expression of the two PPARalpha target genes acyl-CoA oxidase and liver FA-binding protein for alpha-methyl TTA, alpha-methyl EPA, and alpha-methyl DHA. Decreased or altered metabolism of these derivatives in the cells cannot be excluded. In conclusion, saturated FA with sulfur in the beta-position and increasing carbon chain length from C9-S acetic acid to C14-S acetic acid have increasing effects as activators of PPARalpha and -gamma in transfection assays. Furthermore, alpha-methyl FA derivatives of a saturated natural FA (palmitic acid), a sulfur-substituted FA (TTA), and PUFA (EPA, DHA, c9,t11 CLA, and t10,c12 CLA) are stronger PPARalpha activators than the unmethylated compounds.

Liver X receptors as insulin-mediating factors in fatty acid and cholesterol biosynthesis.

The nuclear receptor liver X receptor (LXR) alpha, an important regulator of cholesterol and bile acid metabolism, was analyzed after insulin stimulation in liver in vitro and in vivo. A time- and dose-dependent increase in LXRalpha steady-state mRNA level was seen after insulin stimulation of primary rat hepatocytes in culture. A maximal induction of 10-fold was obtained when hepatocytes were exposed to 400 nm insulin for 24 h. Cycloheximide, a potent inhibitor of protein synthesis, prevented induction of LXRalpha mRNA expression by insulin, indicating that the induction is dependent on de novo synthesis of proteins. Stabilization studies using actinomycin D indicated that insulin stimulation increased the half-life of LXRalpha transcripts in cultured primary hepatocytes. Complementary studies where rats and mice were injected with insulin induced LXRalpha mRNA levels and confirmed our in vitro studies. Furthermore, deletion of both the LXRalpha and LXRbeta genes (double knockout) in mice markedly suppressed insulin-mediated induction of an entire class of enzymes involved in both fatty acid and cholesterol metabolism. The discovery of insulin regulation of LXR in hepatic tissue as well as gene targeting studies in mice provide strong evidence that LXRs plays a central role not only in cholesterol homeostasis, but also in fatty acid metabolism. Furthermore, LXRs appear to be important insulin-mediating factors in regulation of lipogenesis.