Tissue-specific autoregulation of the LXRalpha gene facilitates induction of apoE in mouse adipose tissue

The functions of the liver X receptors (LXRs) are not well documented in adipose tissue. We demonstrate here that expression of the LXRalpha gene is highly induced in vivo and in vitro in mouse and human adipocytes in the presence of the synthetic LXR agonist T0901317. This autoregulation is caused by an identified LXR-responsive element motif in the mouse LXRalpha promoter, which is conserved in the human LXRalpha promoter. Using different LXR-deficient mice, we demonstrate that the basal expression level of LXRalpha is increased in LXRbeta(-/-) mice, whereas the basal expression level of LXRbeta is unchanged in LXRalpha(-/-) mice. The two LXRs can compensate for each other in mediating ligand-activated regulation of LXR target genes involved in lipid homeostasis in adipose tissue. Sterol regulatory element binding protein-1 (SREBP-1), ATP binding cassette transporter A1 (ABCA1), ABCG1, as well as apolipoprotein E (apoE) are induced in vivo by T0901317 in wild-type, LXRalpha(-/-) or LXRbeta(-/-) mice but not in LXRalpha(-/-)beta(-/-) mice. Although SREBP-1 and ABCG1 are induced in liver, muscle, and adipose tissue, the apoE, glucose transporter-4 (GLUT4), and LXRalpha genes are specifically induced only in adipose tissue. We suggest that an important aspect of LXRalpha autoregulation in adipose tissue may be to increase the level of LXRalpha over a threshold level necessary to induce the expression of certain target genes.     

Induction of intestinal ATP-binding cassette transporters by a phytosterol-derived liver X receptor agonist

The nuclear receptors liver X receptor (LXR) alpha and LXRbeta serve as oxysterol receptors and regulate the expression of genes involved in lipid metabolism. LXR activation induces the expression of ATP-binding cassette (ABC) transporters, such as ABCG5 and ABCG8, which inhibit intestinal absorption of cholesterol and phytosterols. Although several synthetic LXR agonists have been generated, these compounds have limited clinical application, because they cause hypertriglycemia by inducing the expression of lipogenic genes in the liver. We synthesized derivatives of phytosterols and found some of them to act as LXR agonists. Among them, YT-32 [(22E)-ergost-22-ene-1alpha,3beta-diol], which is related to ergosterol and brassicasterol, is the most potent LXR agonist. YT-32 directly bound to LXRalpha and LXRbeta and induced the interaction of LXRalpha with cofactors, such as steroid receptor coactivator-1, as effectively as the natural ligands, 22(R)-hydroxycholesterol and 24(S),25-epoxycholesterol. Although the nonsteroidal synthetic LXR agonist T0901317 induced the expression of intestinal ABC transporters and liver lipogenic genes, oral administration of YT-32 selectively activated intestinal ABC transporters in mice. Unlike T0901317 treatment, YT-32 inhibited intestinal cholesterol absorption without increasing plasma triglyceride levels. The phytosterol-derived LXR agonist YT-32 might selectively modulate intestinal cholesterol metabolism.     

On the role of liver X receptors in lipid accumulation in adipocytes

The pivotal role of liver X receptors (LXRs) in the metabolic conversion of cholesterol to bile acids in mice is well established. More recently, the LXRalpha promoter has been shown to be under tight regulation by peroxisome proliferator-activated receptors (PPARs), implying a role for LXRalpha in mediating the interplay between cholesterol and fatty acid metabolism. We have studied the role of LXR in fat cells and demonstrate that LXR is regulated during adipogenesis and augments fat accumulation in mature adipocytes. LXRalpha expression in murine 3T3-L1 adipocytes as well as in human adipocytes was up-regulated in response to PPARgamma agonists. Administration of a PPARgamma agonist to obese Zucker rats also led to increased LXRalpha mRNA expression in adipose tissue in vivo. LXR agonist treatment of differentiating adipocytes led to increased lipid accumulation. An increase of the expression of the LXR target genes, sterol regulatory binding protein-1 and fatty acid synthase, was observed both in vivo and in vitro after treatment with LXR agonists for 24 h. Finally, we demonstrate that fat depots in LXRalpha/beta-deficient mice are smaller than in age-matched wild-type littermates. These findings imply a role for LXR in controlling lipid storage capacity in mature adipocytes and point to an intriguing physiological interplay between LXR and PPARgamma in controlling pathways in lipid handling.     

Dissection of the insulin-sensitizing effect of liver X receptor ligands

The liver X receptors (LXRalpha and beta) are nuclear receptors that coordinate carbohydrate and lipid metabolism. Treatment of insulin-resistant mice with synthetic LXR ligands enhances glucose tolerance, inducing changes in gene expression expected to decrease hepatic gluconeogenesis (via indirect suppression of gluconeogenic enzymes) and increase peripheral glucose disposal (via direct up-regulation of glut4 in fat). To evaluate the relative contribution of each of these effects on whole-body insulin sensitivity, we performed hyperinsulinemic-euglycemic clamps in high-fat-fed insulin-resistant rats treated with an LXR agonist or a peroxisome proliferator-activated receptor gamma ligand. Both groups showed significant improvement in insulin action. Interestingly, rats treated with LXR ligand had lower body weight and smaller fat cells than controls. Insulin-stimulated suppression of the rate of glucose appearance (Ra) was pronounced in LXR-treated rats, but treatment failed to enhance peripheral glucose uptake (R'g), despite increased expression of glut4 in epididymal fat. To ascertain whether LXR ligands suppress hepatic gluconeogenesis directly, mice lacking LXRalpha (the primary isotype in liver) were treated with LXR ligand, and gluconeogenic gene expression was assessed. LXR activation decreased expression of gluconeogenic genes in wild-type and LXRbeta null mice, but failed to do so in animals lacking LXRalpha. Our observations indicate that despite inducing suggestive gene expression changes in adipose tissue in this model of diet-induced insulin resistance, the antidiabetic effect of LXR ligands is primarily due to effects in the liver that appear to require LXRalpha. These findings have important implications for clinical development of LXR agonists as insulin sensitizers.     

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.     

Multiple roles of the nuclear receptors for oxysterols liver X receptor to maintain male fertility

Oxysterol nuclear receptors liver X receptor (LXR)alpha and LXRbeta are known to regulate lipid homeostasis in cells exposed to high amounts of cholesterol and/or fatty acids. In order to elucidate the specific and redundant roles of the LXRs in the testis, we explored the reproductive phenotypes of mice deficient of LXRalpha, LXRbeta, and both, of which only the lxralpha;beta-/- mice are infertile by 5 months of age. We demonstrate that LXRalpha-deficient mice had lower levels of testicular testosterone that correlated with a higher apoptotic rate of the germ cells. LXRbeta-deficient mice showed increased lipid accumulation in the Sertoli cells and a lower proliferation rate of the germ cells. In lxralpha;beta-/- mice, fatty acid metabolism was affected through a decrease of srebp1c and increase in scd1 mRNA expression. The retinoid acid signaling pathway was also altered in lxralpha;beta-/- mice, with a higher accumulation of all-trans retinoid receptor alpha, all-trans retinoid receptor beta, and retinoic aldehyde dehydrogenase-2 mRNA. Combination of these alterations might explain the deleterious phenotype of infertility observed only in lxralpha;beta-/- mice, even though lipid homeostasis seemed to be first altered. Wild-type mice treated with a specific LXR agonist showed an increase of testosterone production involving both LXR isoforms. Altogether, these data identify new roles of each LXR, collaborating to maintain both integrity and functions of the testis.     

Further modification on phenyl acetic acid based quinolines as liver X receptor modulators

A series of phenyl acetic acid based quinolines was prepared as LXR modulators. An SAR study in which the C-3 and C-8 positions of the quinoline core were varied led to the identification of two potent LXR agonists 23 and 27. Both compounds displayed good binding affinity for LXRbeta and LXRalpha, and increased expression of ABCA1 in THP-1 cells. These two compounds also had desirable pharmacokinetic profiles in mice and displayed in vivo efficacy in a 12-week Apo E knockout mouse lesion model.     

TSH signaling and cell survival in 3T3-L1 preadipocytes

Thyroid-stimulating hormone (TSH) action in adipose tissue remains largely unknown. Our previous work indicates that human preadipocytes express functional TSH receptor (TSHR) protein, demonstrated by TSH activation of p70 S6 kinase (p70 S6K). We have now studied murine 3T3-L1 preadipocytes to further characterize TSH signaling and cellular action. Western blot analysis of 3T3-L1 preadipocyte lysate revealed the 100-kDa mature processed form of TSHR. TSH activated p70 S6K and protein kinase B (PKB/Akt), as measured by immunoblot analysis. Preincubation with wortmannin or LY-294002 completely blocked TSH activation of p70 S6K and PKB/Akt, implicating phosphoinositide 3-kinase (PI3K) in their regulation. TSH increased phosphotyrosine protein(s) in the 125-kDa region and augmented the associated PI3K activity fourfold. TSH had no effect on cAMP levels in 3T3-L1 preadipocytes, suggesting that adenylyl cyclase is not involved in TSH activation of the PI3K-PKB/Akt-p70 S6K pathway. 3T3-L1 preadipocyte cell death was reduced by 29-76% in serum-deprived (6 h) preadipocytes treated with 1-20 microM TSH. In the presence of 20 microM TSH, an 88% reduction in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-positive cells was observed in serum-starved (3 h) 3T3-L1 preadipocytes as well as a 93% reduction in the level of cleaved activated caspase 3. In summary, TSH acts as a survival factor in 3T3-L1 preadipocytes. TSH does not stimulate cAMP accumulation in these cells but instead activates a PI3K-PKB/Akt-p70 S6K pathway.     

Niemann-Pick C1 like 1 gene expression is down-regulated by LXR activators in the intestine

Niemann-Pick C1 like 1 (NPC1L1) is a protein critical for intestinal cholesterol absorption. The nuclear receptors peroxisome proliferator-activated receptor alpha (PPARalpha) and liver X receptors (LXRalpha and LXRbeta) are major regulators of cholesterol homeostasis and their activation results in a reduced absorption of intestinal cholesterol. The goal of this study was to define the role of PPARalpha and LXR nuclear receptors in the regulation of NPC1L1 gene expression. We show that LXR activators down-regulate NPC1L1 mRNA levels in the human enterocyte cell line Caco-2/TC7, whereas PPARalpha ligands have no effect. Furthermore, NPC1L1 mRNA levels are decreased in vivo, in duodenum of mice treated with the LXR agonist T0901317. In conclusion, the present study identifies NPC1L1 as a novel LXR target gene further supporting a crucial role of LXR in intestinal cholesterol homeostasis.