Nogo-B receptor deficiency increases liver X receptor alpha nuclear translocation and hepatic lipogenesis through an adenosine monophosphate-activated protein kinase alpha-dependent pathway.
ABSTRACT: Nogo-B receptor (NgBR) was identified as a specific receptor for binding Nogo-B and is essential for the stability of Niemann-Pick type C2 protein (NPC2) and NPC2-dependent cholesterol trafficking. Here, we report that NgBR expression levels decrease in the fatty liver and that NgBR plays previously unrecognized roles in regulating hepatic lipogenesis through NPC2-independent pathways. To further elucidate the pathophysiological role of NgBR in mammals, we generated NgBR liver-specific knockout mice and investigated the roles of NgBR in hepatic lipid homeostasis. The results showed that NgBR knockout in mouse liver did not decrease NPC2 levels or increase NPC2-dependent intracellular cholesterol levels. However, NgBR deficiency still resulted in remarkable cellular lipid accumulation that was associated with increased free fatty acids and triglycerides in hepatocytes in vitro and in mouse livers in vivo. Mechanistically, NgBR deficiency specifically promotes the nuclear translocation of the liver X receptor alpha (LXR?) and increases the expression of LXR?-targeted lipogenic genes. LXR? knockout attenuates the accumulation of free fatty acids and triglycerides caused by NgBR deficiency. In addition, we elucidated the mechanisms by which NgBR bridges the adenosine monophosphate-activated protein kinase alpha signaling pathway with LXR? nuclear translocation and LXR?-mediated lipogenesis.NgBR is a specific negative regulator for LXR?-dependent hepatic lipogenesis. Loss of NgBR may be a potential trigger for inducing hepatic steatosis. (Hepatology 2016;64:1559-1576).
Project description:Cellular mechanisms that mediate steatohepatitis, an increasingly prevalent condition in the Western world for which no therapies are available, are poorly understood. Despite the fact that its synthetic agonists induce fatty liver, the liver X receptor (LXR) transcription factor remains a target of interest because of its anti-atherogenic, cholesterol removal, and anti-inflammatory activities. Here we show that tetratricopeptide repeat domain protein 39B (Ttc39b, C9orf52) (T39), a high-density lipoprotein gene discovered in human genome-wide association studies, promotes the ubiquitination and degradation of LXR. Chow-fed mice lacking T39 (T39(-/-)) display increased high-density lipoprotein cholesterol levels associated with increased enterocyte ATP-binding cassette transporter A1 (Abca1) expression and increased LXR protein without change in LXR messenger RNA. When challenged with a high fat/high cholesterol/bile salt diet, T39(-/-) mice or mice with hepatocyte-specific T39 deficiency show increased hepatic LXR protein and target gene expression, and unexpectedly protection from steatohepatitis and death. Mice fed a Western-type diet and lacking low-density lipoprotein receptor (Ldlr(-/-)T39(-/-)) show decreased fatty liver, increased high-density lipoprotein, decreased low-density lipoprotein, and reduced atherosclerosis. In addition to increasing hepatic Abcg5/8 expression and limiting dietary cholesterol absorption, T39 deficiency inhibits hepatic sterol regulatory element-binding protein 1 (SREBP-1, ADD1) processing. This is explained by an increase in microsomal phospholipids containing polyunsaturated fatty acids, linked to an LXR?-dependent increase in expression of enzymes mediating phosphatidylcholine biosynthesis and incorporation of polyunsaturated fatty acids into phospholipids. The preservation of endogenous LXR protein activates a beneficial profile of gene expression that promotes cholesterol removal and inhibits lipogenesis. T39 inhibition could be an effective strategy for reducing both steatohepatitis and atherosclerosis.
Project description:Liver X receptors (LXR?/?) and carbohydrate response element-binding proteins (ChREBP?/?) are key players in the transcriptional control of hepatic <i>de novo</i> lipogenesis. LXR?/? double knockout (LXR?<sup>-/-</sup>/?<sup>-/-</sup>) mice have reduced feeding-induced nuclear <i>O</i>-linked <i>N</i>-acetylglucosamine (<i>O</i>-GlcNAc) signaling, ChREBP? activity, and lipogenic gene expression in livers, suggesting important roles for LXRs in linking hepatic glucose utilization to lipid synthesis. However, the role of LXRs in fructose-induced ChREBP activation and lipogenesis is currently unknown. In this study, we studied the effects of high fructose or high glucose feeding on hepatic carbohydrate metabolism and lipogenic gene expression in livers from fasted (24 h) and fasted-refed (12 h) wild type and LXR? knockout (LXR?<sup>-/-</sup>) mice. Hepatic lipogenic gene expression was reduced in glucose fed, but not fructose fed LXR?<sup>-/-</sup> mice. This was associated with lower expression of liver pyruvate-kinase (<i>L-pk</i>) and <i>Chrebp?</i>, indicating reduced ChREBP? activity in glucose fed, but not fructose fed mice. Interestingly, ChREBP binding to the <i>L-pk</i> promoter was increased in fructose fed LXR?<sup>-/-</sup> mice, concomitant with increased glucose-6-phosphatase (<i>G6pc</i>) expression and <i>O</i>-GlcNAc modified LXR?, suggesting a role for LXR? in regulating ChREBP? activity upon fructose feeding. In conclusion, we propose that LXR? is an important regulator of hepatic lipogenesis and ChREBP? activity upon glucose, but not fructose feeding in mice.
Project description:Ligand-activated liver X receptor ? (LXR?) upregulates the expression of hepatic lipogenic genes, which leads to triglyceride (TG) accumulation, resulting in nonalcoholic fatty liver disease (NAFLD). Thus, LXR? regulation may provide a novel therapeutic target against NAFLD. However, histone methylation-mediated epigenetic regulation involved in LXR?-dependent lipogenesis is poorly understood. In this study, we investigated the functional role of the histone demethylase Jumonji domain-containing protein 2B (JMJD2B) in LXR?-dependent lipogenesis. JMJD2B expression level was upregulated in HepG2 cells treated with LXR? agonist T0901317 or palmitate and the liver of mice administered with T0901317 or fed a high-fat diet. Knockdown of JMJD2B using siRNA abrogated T0901317-induced LXR?-dependent lipogenic gene expression and lowered intracellular TG accumulation. Conversely, overexpression of JMJD2B in HepG2 cells upregulated the expression of LXR?-dependent lipogenic genes, in line with increased intracellular TG levels. JMJD2B overexpression or T0901317 treatment induced the recruitment of JMJD2B and LXR? to LXR response elements (LXRE) in the promoter region of LXR?-target gene and reduced the enrichment of H3K9me2 and H3K9me3 in the vicinity of the LXRE. Furthermore, JMJD2B enhanced T0901317 or LXR?-induced transcriptional activities of reporters containing LXRE. A co-immunoprecipitation assay revealed that JMJD2B interacted with activated LXR?. Moreover, overexpression of JMJD2B in mice resulted in upregulation of hepatic LXR?-dependent lipogenic genes, consistent with development of hepatic steatosis. Taken together, these results indicate that JMJD2B plays a role in LXR?-mediated lipogenesis via removing the repressive histone marks, H3K9me2 and H3K9me3, at LXRE, which might contribute to hepatic steatosis.
Project description:Nogo-B receptor (NgBR) was identified as a receptor specific for Nogo-B. Our previous work has shown that Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro and intersomitic vessel formation via Akt pathway in zebrafish. Here, we further demonstrated the roles of NgBR in regulating vasculature development in mouse embryo and primitive blood vessel formation in embryoid body culture systems, respectively. Our results showed that NgBR homozygous knockout mice are embryonically lethal at E7.5 or earlier, and Tie2Cre-mediated endothelial cell-specific NgBR knockout (NgBR ecKO) mice die at E11.5 and have severe blood vessel assembly defects in embryo. In addition, mutant embryos exhibit dilation of cerebral blood vessel, resulting in thin-walled endothelial caverns. The similar vascular defects also were detected in Cdh5(PAC)-CreERT2 NgBR inducible ecKO mice. Murine NgBR gene-targeting embryonic stem cells (ESC) were generated by homologous recombination approaches. Homozygous knockout of NgBR in ESC results in cell apoptosis. Heterozygous knockout of NgBR does not affect ESC cell survival, but reduces the formation and branching of primitive blood vessels in embryoid body culture systems. Mechanistically, NgBR knockdown not only decreases both Nogo-B and VEGF-stimulated endothelial cell migration by abolishing Akt phosphorylation, but also decreases the expression of CCM1 and CCM2 proteins. Furthermore, we performed immunofluorescence (IF) staining of NgBR in human cerebral cavernous malformation patient tissue sections. The quantitative analysis results showed that NgBR expression levels in CD31 positive endothelial cells is significantly decreased in patient tissue sections. These results suggest that NgBR may be one of important genes coordinating the cerebral vasculature development.
Project description:Hepatic lipogenesis is aberrantly induced in nonalcoholic fatty liver disease (NAFLD) via activation of the LXR-SREBP1c pathway. To date, a number of protein factors impinging on the transcriptional activity of LXR and SREBP1c have been elucidated. However, whether this regulatory axis interfaces with long noncoding RNAs (lncRNAs) remains largely unexplored. Here we show that hepatic expression of the lncRNA Blnc1 is strongly elevated in obesity and NAFLD in mice. Blnc1 is required for the induction of SREBP1c and hepatic lipogenic genes in response to LXR activation. Liver-specific inactivation of Blnc1 abrogates high-fat diet-induced hepatic steatosis and insulin resistance and protects mice from diet-induced nonalcoholic steatohepatitis. Proteomic analysis of the Blnc1 ribonucleoprotein complex identified EDF1 as a component of the LXR transcriptional complex that acts in concert with Blnc1 to activate the lipogenic gene program. These findings illustrate a lncRNA transcriptional checkpoint that licenses excess hepatic lipogenesis to exacerbate insulin resistance and NAFLD.
Project description:HIV patients develop hepatic steatosis. We investigated hepatic steatosis in transgenic mice expressing the HIV-1 accessory protein Vpr (Vpr-Tg) in liver and adipose tissues, and WT mice infused with synthetic Vpr. Vpr-Tg mice developed increased liver triglyceride content and elevated ALT, bilirubin and alkaline phosphatase due to three hepatic defects: 1.6-fold accelerated de novo lipogenesis (DNL), 45% slower fatty acid ß-oxidation, and 40% decreased VLDL-triglyceride export. Accelerated hepatic DNL was due to coactivation by Vpr of liver X receptor-? (LXR?) with increased expression of its lipogenic targets Srebp1c, Chrebp, Lpk, Dgat, Fasn and Scd1, and intranuclear SREBP1c and ChREBP. Vpr enhanced association of LXR? with Lxr? and Srebp1c promoters, increased LXRE-LXR? binding, and broadly altered hepatic expression of LXR?-regulated lipid metabolic genes. Diminished hepatic fatty acid ß-oxidation was associated with decreased mRNA expression of Ppar? and its targets Cpt1, Aox, Lcad, Ehhadh, Hsd10 and Acaa2, and blunted VLDL export with decreased expression of Mttp and its product microsomal triglyceride transfer protein. With our previous findings that Vpr circulates in HIV patients (including those with undetectable plasma HIV-1 RNA), co-regulates the glucocorticoid receptor and PPAR? and transduces hepatocytes, these data indicate a potential role for Vpr in HIV-associated fatty liver disease.
Project description:Previous findings supported that fetuin B, a new hepatokine, may be involved in the development of hepatic steatosis, but the mechanism is still unknown. This study aims to investigate the role of fetuin B in hepatic steatosis in C57BL/6 mice and HepG2 cells. 1) We found that the administration of recombinant fetuin B aggravated hepatic lipid accumulation caused by free fatty acids (FFAs) or high fat diet, in vivo and in vitro. It lowered the phosphorylated AMPK levels and activated the LXR-SREBP1c pathway, accompanied by the downregulation of fatty acid oxidation and upregulation of lipogenesis. Furthermore, in HepG2 cells exposed to recombinant fetuin B and FFAs, the AMPK agonist depressed the LXR-SREBP1c pathway and alleviated lipid accumulation. The knockdown of LXR protected against steatosis but failed to change phosphorylated AMPK. 2) The knockdown of fetuin B by siRNA or shRNA alleviated lipid accumulation, in vivo and in vitro. It enhanced phosphorylated AMPK and depressed the LXR-SREBP1c pathway, accompanied by upregulation of fatty acid oxidation and downregulation of lipogenesis. Moreover, in HepG2 cells exposed to fetuin B siRNA and FFAs, LXR agonist aggravated lipid accumulation but failed to influence AMPK. This study indicated that fetuin B aggravated LXR-mediated hepatic steatosis through AMPK. It might offer new insights into clinical management and biomarker research on fatty liver.
Project description:Excessive intake of fat causes accumulation of fat in liver, leading to non-alcoholic fatty liver disease (NAFLD). High-fat diet (HFD) upregulates the expression of Factor D, a complement pathway component, in the liver of mice. However, the functions of Factor D in liver are not well known. Therefore, the current study investigated the relationship between Factor D and hepatic lipid accumulation using CRISPR/Cas9-mediated Factor D knockout (FD-KO) mice. Factor D deficiency downregulated expression of genes related to fatty acid uptake and de novo lipogenesis in the liver. Furthermore, Factor D deficiency reduced the expression of inflammatory factors (Tnf and Ccl2) and fibrosis markers and decreased accumulation of F4/80-positive macrophages. These data suggest that the Factor D deficiency improved hepatic lipid accumulation and hepatic inflammation in HFD-fed mice.
Project description:NgBR is a transmembrane protein identified as a Nogo-B-interacting protein and recently has been shown to be a subunit required for cis-prenyltransferase (cisPTase) activity. To investigate the integrated role of NgBR in vascular development, we have characterized endothelial-specific NgBR knockout embryos. Here, we show that endothelial-specific NgBR knockout results in embryonic lethality due to vascular development defects in yolk sac and embryo proper. Loss of NgBR in endothelial cells reduces proliferation and promotes apoptosis of the cells largely through defects in the glycosylation of key endothelial proteins including VEGFR2, VE-cadherin, and CD31, and defective glycosylation can be rescued by treatment with the end product of cisPTase activity, dolichol phosphate. Moreover, NgBR functions in endothelial cells during embryogenesis are Nogo-B independent. These data uniquely show the importance of NgBR and protein glycosylation during vascular development.
Project description:Olive oil consumption is beneficial for health as it is associated with a decreased prevalence of cancer and cardiovascular diseases. Oleic acid is, by far, the most abundant component of olive oil. Since it can be made through de novo synthesis in animals, it is not an essential fatty acid. While it has become clear that dietary oleic acid regulates many biological processes, the signaling pathway involved in these regulations remains poorly defined. In this work we tested the impact of an oleic acid-rich diet on hepatic gene expression. We were particularly interested in addressing the contribution of Liver X Receptors (LXR) in the control of genes involved in hepatic lipogenesis, an essential process in whole body energy homeostasis. We used wild-type mice and transgenic mice deficient for both ? and ? Liver X Receptor isoforms (LXR-/-) fed a control or an oleate enriched diet. We observed that hepatic-lipid accumulation was enhanced as well as the expression of lipogenic genes in the liver of wild-type mice fed the oleate enriched diet. In contrast, none of these changes occurred in the liver of LXR-/- mice. Strikingly, oleate-rich diet reduced cholesterolemia in wild-type mice and induced signs of liver inflammation and damage in LXR-/- mice but not in wild-type mice. This work suggests that dietary oleic acid reduces cholesterolemia while promoting LXR-dependent hepatic lipogenesis without detrimental effects to the liver.