Hilde I. Nebb

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

The nuclear receptor liver X receptor (LXR) α, an important regulator of cholesterol and bile acid metabolism, was analyzed after insulin stimulation in liver in vitro andin vivo. A time- and dose-dependent increase in LXRα 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 nminsulin for 24 h. Cycloheximide, a potent inhibitor of protein synthesis, prevented induction of LXRα 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 LXRα transcripts in cultured primary hepatocytes. Complementary studies where rats and mice were injected with insulin induced LXRα mRNA levels and confirmed our in vitrostudies. Furthermore, deletion of both the LXRα and LXRβ 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.

Genome-Wide Profiling of Liver X Receptor, Retinoid X Receptor, and Peroxisome Proliferator-Activated Receptor α in Mouse Liver Reveals Extensive Sharing of Binding Sites

ABSTRACT The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR) signaling pathways, and subsequent chromatin immunoprecipitation-sequencing (ChIP-seq) mapping of PPARα binding demonstrated binding of PPARα to 71 to 88% of the identified LXR-RXR binding sites. The combination of sequence analysis of shared binding regions and sequential ChIP on selected sites indicate that LXR-RXR and PPARα-RXR bind to degenerate response elements in a mutually exclusive manner. Together, our findings suggest extensive and unexpected cross talk between hepatic LXR and PPARα at the level of binding to shared genomic sites.

Expression of the insulin-responsive glucose transporter GLUT4 in adipocytes is dependent on liver x receptor α

The insulin-responsive glucose transporter GLUT4 plays a crucial role in insulin-mediated facilitated glucose uptake into adipose tissue and muscle, and impaired expression of GLUT4 has been linked to obesity and diabetes. In this study, we demonstrate that liver X receptors (LXRs) regulate the expression of GLUT4 through direct interaction with a conserved LXR response element in the GLUT4 promoter. The expression of GLUT4 in WAT is induced by a potent LXR agonist in wild type, LXRα-/-, and LXRβ-/- mice but not in LXRα-/-β-/- mice, demonstrating that both LXRs are able to mediate ligand activated transcription of the GLUT4 gene. However, basal and insulin stimulated expression of GLUT4 in epididymal WAT is reduced only in mice carrying ablation of the LXRα isoform. The expression of GLUT4 is furthermore correlated to the induction of LXRα during mouse and human adipocyte differentiation. LXRβ is thus apparently not able to rescue basal expression of GLUT4 in the absence of LXRα. We have previously demonstrated that LXRα is down-regulated in animal models of obesity and diabetes, thus revealing a striking correlation between GLUT4 and LXRα expression in insulin-resistant conditions. This suggests that the LXRα isoform has a unique role in adipose expression of GLUT4 and suggests that alteration of adipose tissue expression of LXRα might be a novel tool to normalize the expression of a gene that is dysregulated in diabetic and insulin-resistant conditions.

Trans10, cis12-conjugated linoleic acid prevents triacylglycerol accumulation in adipocytes by acting as a PPARγ modulator

A group of polyunsaturated fatty acids called conjugated linoleic acids (CLAs) are found in ruminant products, where the most common isomers are cis9, trans11 (c 9,t11) and trans10, cis12 (t10,c12) CLA. A crude mixture of these isomers has been shown in animal studies to alter body composition by a reduction in body fat mass as well as an increase in lean body mass, with the t10,c12 isomer having the most pronounced effect. The objective of this study was to establish the molecular mechanisms by which t10,c12 CLA affects lipid accumulation in adipocytes. We have shown that t10,c12 CLA prevents lipid accumulation in human and mouse adipocytes at concentrations as low as 5 μM and 25 μM, respectively. t10,c12 CLA fails to activate peroxisome proliferator-activated receptor γ (PPARγ) but selectively inhibits thiazolidinedione-induced PPARγ activation in 3T3-L1 adipocytes. Treatment of mature adipocytes with t10,c12 CLA alone or in combination with Darglitazone down-regulates the mRNA expression of PPARγ as well as its target genes, fatty acid binding protein (aP2) and liver X receptor α (LXRα). Taken together, our results suggest that the trans10, cis12 CLA isomer prevents lipid accumulation in adipocytes by acting as a PPARγ modulator.

PPARα activators and fasting induce the expression of adipose differentiation-related protein in liver

The adipose differentiation-related protein (ADFP)/adipophilin belongs to a family of PAT (for perilipin, ADFP, and TIP47) proteins that associate on the surface of lipid droplets (LDs). Except for LD association, a clear role for ADFP has not been found. We demonstrate that ADFP is transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα) in mouse liver and rat and human hepatoma cells through a highly conserved direct repeat-1(DR-1) element. Although the ADFP mRNA is highly increased by a synthetic PPARα agonist, the ADFP protein is only substantially increased in cells containing LDs, such as hepatocytes incubated with fatty acids, and in livers of fasted mice. ADFP is induced by fasting even in the absence of a functional PPARα, in marked contrast to the PPARα target gene acyl-coenzyme A oxidase-1. Activation of LXRs, which stimulates LD formation through the activation of lipogenesis, does not affect ADFP mRNA levels. TIP47, another PAT member known to be expressed in liver, was unaffected by all treatments. This constitutively expressed PAT member seems to be less transcriptionally regulated than ADFP. These observations suggest that ADFP is primarily a fasting-induced protein in liver that coats the newly synthesized triacylglycerol-containing LDs formed during fasting.

Tissue-specific autoregulation of the LXRα 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 LXRα 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 LXRα promoter, which is conserved in the human LXRα promoter. Using different LXR-deficient mice, we demonstrate that the basal expression level of LXRα is increased in LXRβ−/− mice, whereas the basal expression level of LXRβ is unchanged in LXRα−/− 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, LXRα−/− or LXRβ−/− mice but not in LXRα−/−β−/− mice. Although SREBP-1 and ABCG1 are induced in liver, muscle, and adipose tissue, the apoE, glucose transporter-4 (GLUT4), and LXRα genes are specifically induced only in adipose tissue. We suggest that an important aspect of LXRα autoregulation in adipose tissue may be to increase the level of LXRα over a threshold level necessary to induce the expression of certain target genes.

Regulation of hepatic fatty acid elongase 5 by LXRα–SREBP-1c

Dietary essential fatty acids linoleic acid and alpha-linolenic acid are converted to arachidonic-, eicosapentaenoic-, and docosahexaenoic acid under tight regulation by nutritional status and hormones. Hepatic fatty acid elongase 5 (Elovl5) elongates C18-20 polyunsaturated fatty acids (PUFAs) and is important for biosynthesis of C20-22 PUFAs. We demonstrate that Liver X Receptor alpha (LXRalpha) and sterol regulatory binding protein-1c (SREBP-1c) regulate hepatic Elovl5 expression. LXRalpha and LXRbeta play different roles in maintenance of basal expression of Elovl5. LXRalpha is necessary for basal as well as LXR agonist induced Elovl5 transcription. Promoter studies revealed that the mouse Elovl5 gene is a direct SREBP-1c target. The up-regulation of Elovl5 expression by LXR agonist is likely secondary to the induction of SREBP-1c. PUFAs repress expression of SREBP-1c and Elovl5, but when combined with LXR ligand stimulation, which increases SREBP-1c mRNA and nuclear SREBP-1c, Elovl5 mRNA levels are restored to normal. Our studies suggest that an LXRalpha-SREBP-1c pathway plays a regulatory role in hepatic biosynthesis of PUFAs through transcriptional activation of Elovl5 as well as other desaturases. The stimulatory role of LXRalpha-SREBP-1c in the production of PUFAs enables the possibility for a feedback regulation of hepatic lipogenesis through PUFA mediated repression of SREBP-1c expression.

Intracellular Nicotinamide Phosphoribosyltransferase Protects against Hepatocyte Apoptosis and Is Down-Regulated in Nonalcoholic Fatty Liver Disease

CONTEXT Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in Western and non-Western countries, but its pathogenesis is not fully understood. OBJECTIVE Based on the role of nicotinamide phosphoribosyltransferase (NAMPT) in fat and glucose metabolism and cell survival, we hypothesized a role for NAMPT/visfatin in the pathogenesis of NAFLD-related disease. DESIGN AND SETTING We conducted clinical studies at a referral medical center in well-characterized NAFLD patients (n = 58) and healthy controls (n = 27). In addition we performed experimental in vitro studies in hepatocytes. MAIN OUTCOME MEASURES We examined 1) the hepatic and systemic expression of NAMPT/visfatin in patients with NAFLD and control subjects, 2) the hepatic regulation of NAMPT/visfatin, and 3) the effect of NAMPT/visfatin on hepatocyte apoptosis. RESULTS Our main findings were as follows. 1) Patients with NAFLD had decreased NAMPT/visfatin expression both systemically in serum and within the hepatic tissue, with no difference between simple steatosis and nonalcoholic steatohepatitis. 2) By studying the hepatic regulation of NAMPT/visfatin in wild-type and peroxisome proliferators-activated receptor (PPAR)alpha(-/-) mice as well as in hepatocytes, we showed that PPARalpha activation and glucose may be involved in the down-regulation of hepatic NAMPT/visfatin expression in NAFLD. 4) Within the liver, NAMPT/visfatin was located to hepatocytes, and our in vitro studies showed that NAMPT/visfatin exerts antiapoptotic effects in these cells, involving enzymatic synthesis of nicotinamide adenine dinucleotide. CONCLUSION Based on these findings, we suggest a role for decreased NAMPT/visfatin levels in hepatocyte apoptosis in NAFLD-related disease.

Nuclear Receptor Liver X Receptor Is O-GlcNAc-modified in Response to Glucose

Post-translational modification of nucleocytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has for the last 25 years emerged as an essential glucose-sensing mechanism. The liver X receptors (LXRs) function as nutritional sensors for cholesterol-regulating lipid metabolism, glucose homeostasis, and inflammation. LXRs are shown to be post-translationally modified by phosphorylation, acetylation, and sumoylation, affecting their target gene specificity, stability, and transactivating and transrepressional activity, respectively. In the present study, we show for the first time that LXRα and LXRβ are targets for glucose-hexosamine-derived O-GlcNAc modification in human Huh7 cells. Furthermore, we observed increased hepatic LXRα O-GlcNAcylation in vivo in refed mice and in streptozotocin-induced refed diabetic mice. Importantly, induction of LXRα O-GlcNAcylation in both mouse models was concomitant with increased expression of the lipogenic gene SREBP-1c (sterol regulatory element-binding protein 1c). Furthermore, glucose increased LXR/retinoic acid receptor-dependent activation of luciferase reporter activity driven by the mouse SREBP-1c promoter via the hexosamine biosynthetic pathway in Huh7 cells. Altogether, our results suggest that O-GlcNAcylation of LXR is a novel mechanism by which LXR acts as a glucose sensor affecting LXR-dependent gene expression, substantiating the crucial role of LXR as a nutritional sensor in lipid and glucose metabolism.

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.