micro RNA ‐379 couples glucocorticoid hormones to dysfunctional lipid homeostasis
2014; Springer Nature; Volume: 34; Issue: 3 Linguagem: Inglês
10.15252/embj.201490464
ISSN1460-2075
AutoresRoldan M. de Guia, Adam J. Rose, Anke Sommerfeld, Oksana Seibert, Daniela Strzoda, Annika Zota, Yvonne Feuchter, Anja Krones‐Herzig, Tjeerd Sijmonsma, Milen Kirilov, Carsten Sticht, Norbert Gretz, Geesje M. Dallinga‐Thie, Sven Diederichs, Nora Klöting, Matthias Blüher, Mauricio Berriel Díaz, Stephan Herzig,
Tópico(s)RNA Research and Splicing
ResumoArticle15 December 2014free access microRNA-379 couples glucocorticoid hormones to dysfunctional lipid homeostasis Roldan M de Guia Roldan M de Guia Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Adam J Rose Adam J Rose Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Anke Sommerfeld Anke Sommerfeld Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Oksana Seibert Oksana Seibert Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Daniela Strzoda Daniela Strzoda Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Annika Zota Annika Zota Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Yvonne Feuchter Yvonne Feuchter Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Anja Krones-Herzig Anja Krones-Herzig Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Tjeerd Sijmonsma Tjeerd Sijmonsma Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Milen Kirilov Milen Kirilov Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Carsten Sticht Carsten Sticht Medical Research Center, Klinikum Mannheim, Mannheim, Germany Search for more papers by this author Norbert Gretz Norbert Gretz Medical Research Center, Klinikum Mannheim, Mannheim, Germany Search for more papers by this author Geesje Dallinga-Thie Geesje Dallinga-Thie Department of Vascular Medicine, AMC Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Sven Diederichs Sven Diederichs Helmholtz-University-Group Molecular RNA Biology and Cancer, DKFZ, Heidelberg, Germany Institute of Pathology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Nora Klöting Nora Klöting Department of Medicine, University of Leipzig, Leipzig, Germany Search for more papers by this author Matthias Blüher Matthias Blüher Department of Medicine, University of Leipzig, Leipzig, Germany Search for more papers by this author Mauricio Berriel Diaz Mauricio Berriel Diaz Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Stephan Herzig Corresponding Author Stephan Herzig Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Roldan M de Guia Roldan M de Guia Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Adam J Rose Adam J Rose Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Anke Sommerfeld Anke Sommerfeld Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Oksana Seibert Oksana Seibert Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Daniela Strzoda Daniela Strzoda Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Annika Zota Annika Zota Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Yvonne Feuchter Yvonne Feuchter Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Anja Krones-Herzig Anja Krones-Herzig Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Tjeerd Sijmonsma Tjeerd Sijmonsma Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Milen Kirilov Milen Kirilov Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Carsten Sticht Carsten Sticht Medical Research Center, Klinikum Mannheim, Mannheim, Germany Search for more papers by this author Norbert Gretz Norbert Gretz Medical Research Center, Klinikum Mannheim, Mannheim, Germany Search for more papers by this author Geesje Dallinga-Thie Geesje Dallinga-Thie Department of Vascular Medicine, AMC Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Sven Diederichs Sven Diederichs Helmholtz-University-Group Molecular RNA Biology and Cancer, DKFZ, Heidelberg, Germany Institute of Pathology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Nora Klöting Nora Klöting Department of Medicine, University of Leipzig, Leipzig, Germany Search for more papers by this author Matthias Blüher Matthias Blüher Department of Medicine, University of Leipzig, Leipzig, Germany Search for more papers by this author Mauricio Berriel Diaz Mauricio Berriel Diaz Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Stephan Herzig Corresponding Author Stephan Herzig Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany Search for more papers by this author Author Information Roldan M Guia1, Adam J Rose1, Anke Sommerfeld1, Oksana Seibert1, Daniela Strzoda1, Annika Zota1, Yvonne Feuchter1, Anja Krones-Herzig1, Tjeerd Sijmonsma1, Milen Kirilov1, Carsten Sticht2, Norbert Gretz2, Geesje Dallinga-Thie3, Sven Diederichs4,5, Nora Klöting6, Matthias Blüher6, Mauricio Berriel Diaz1 and Stephan Herzig 1 1Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance and Network Aging Research, German Cancer Research Center (DKFZ) Heidelberg, Center for Molecular Biology (ZMBH) and University Hospital, Heidelberg University, Heidelberg, Germany 2Medical Research Center, Klinikum Mannheim, Mannheim, Germany 3Department of Vascular Medicine, AMC Amsterdam, Amsterdam, The Netherlands 4Helmholtz-University-Group Molecular RNA Biology and Cancer, DKFZ, Heidelberg, Germany 5Institute of Pathology, Heidelberg University, Heidelberg, Germany 6Department of Medicine, University of Leipzig, Leipzig, Germany *Corresponding author. Tel: +49 6221 42 3594; E-mail: [email protected] The EMBO Journal (2015)34:344-360https://doi.org/10.15252/embj.201490464 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In mammals, glucocorticoids (GCs) and their intracellular receptor, the glucocorticoid receptor (GR), represent critical checkpoints in the endocrine control of energy homeostasis. Indeed, aberrant GC action is linked to severe metabolic stress conditions as seen in Cushing's syndrome, GC therapy and certain components of the Metabolic Syndrome, including obesity and insulin resistance. Here, we identify the hepatic induction of the mammalian conserved microRNA (miR)-379/410 genomic cluster as a key component of GC/GR-driven metabolic dysfunction. Particularly, miR-379 was up-regulated in mouse models of hyperglucocorticoidemia and obesity as well as human liver in a GC/GR-dependent manner. Hepatocyte-specific silencing of miR-379 substantially reduced circulating very-low-density lipoprotein (VLDL)-associated triglyceride (TG) levels in healthy mice and normalized aberrant lipid profiles in metabolically challenged animals, mediated through miR-379 effects on key receptors in hepatic TG re-uptake. As hepatic miR-379 levels were also correlated with GC and TG levels in human obese patients, the identification of a GC/GR-controlled miRNA cluster not only defines a novel layer of hormone-dependent metabolic control but also paves the way to alternative miRNA-based therapeutic approaches in metabolic dysfunction. Synopsis The discovery of miR-379 as a direct glucocorticoid receptor target in the liver integrates miRNAs in the physiological control of lipid homeostasis. The conserved microRNA (miR)-379/410 genomic cluster is a direct GR target. Silencing of miR-379 ameliorates obesity-related hypertriglyceridemia. miR-379 acts through LSR- and LDLR-mediated hepatic lipid uptake. miR-379 levels correlate with serum cortisol and triglycerides in human obese patients. Introduction The hypothalamic–pituitary–adrenal (HPA) endocrine axis is a critical physiological stress circuit to maintain body homeostasis during diverse situations such as trauma, exercise, or nutrient deprivation, mediated in major parts through adrenal glucocorticoid hormone (GC) action (Rose & Herzig, 2013). In metabolic control, GC signaling acts as a major counter-regulatory system against insulin action, and states of either endogenous or exogenous GC deficiency or excess, for example, Addison's disease, Cushing's syndrome, or GC therapy, respectively, are characterized by severe perturbations in systemic energy metabolism that closely mimic aspects of the Metabolic Syndrome (Vegiopoulos & Herzig, 2007). Indeed, aberrantly elevated GC activity and/or levels are discussed to be linked to the major components of the syndrome, including obesity, insulin resistance, hyperglycemia, and systemic dyslipidemia (Anagnostis et al, 2009). GC levels have been found to be elevated in insulin-resistant patients and are strongly associated with a hyperglycemic and fatty liver phenotype (Phillips et al, 1998). In congruence to this, recent clinical trials using inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), the key enzyme in pre-receptor GC generation, have been proven safe and effective in reducing hepatic liver fat content in obese humans (Stefan et al, 2014), suggesting that GC can—at least in part—drive major aspects of metabolic dysfunction as associated with human obesity. At the molecular level, GC action is mediated through the glucocorticoid receptor (GR), a member of the nuclear receptor transcription factor family (Opherk et al, 2004; Lemke et al, 2008). In liver, numerous direct protein-encoding target genes of the GR have been identified by both classical and high-throughput chromatin recruitment studies (Wang et al, 2004), mediating the GC/GR impact on hepatic gluconeogenesis, triglyceride (TG), and bile acid metabolism (Herzig et al, 2001; Lemke et al, 2008: Rose et al, 2011). In contrast to the well-described protein mediators of GC/GR metabolic action, additional layers of the GC/GR-dependent molecular network and mechanisms of metabolic fine-tuning still remain elusive. Recently, a class of small non-coding RNAs (microRNAs, miRs) has emerged as a critical but as-yet largely unexplored layer of metabolic control. Indeed, individual miRNAs have been found to regulate diverse aspects of energy homeostasis, including pancreatic beta-cell insulin secretion, adipose tissue lipid storage, and hepatic cholesterol and lipid handling (Rottiers & Naar, 2012). However, no miRNA targets of the endocrine GC/GR axis in metabolic dysfunction have been identified to date, prompting us to test the hypothesis that distinct miRNAs serve as molecular output effectors of the HPA-GC/GR metabolic control circuit. Results The miR-379/410 cluster represents a direct GR target in liver To define GC/GR-dependent miRNA networks with immediate relevance in metabolic dysfunction, we initially performed large-scale miRNA expression profiling of livers from wild-type and db/db diabetic mice as a model for diabesity-related hyperglucocorticoidemia (Lemke et al, 2008). A total of 36 miRNAs were found to be up- or down-regulated more than twofold between these animals (Fig 1A). Next, we aligned this data set with a second miRNA signature, resulting from differential expression profiling between mice lacking the GR specifically in hepatocytes and controls (Rose et al, 2011). Data cross-comparison revealed a set of 10 miRNAs that showed significant down-regulation in response to GR deficiency. Some of these miRNAs were simultaneously induced in obesity-related diabetes (Fig 1A), indicating that these miRNAs represent bona fide mediators of GC/GR-driven metabolic dysfunction. Intriguingly, 6 out of 10 GC/GR-targeted miRNAs are located in the miR-379/410 genomic cluster that is conserved between mammalian species (Glazov et al, 2008), which resides on mouse and human chromosomes 12 and 14, respectively. Indeed, selective expression analysis verified the induction of five cluster members in db/db as well as in mice chronically treated with the GC analogue dexamethasone (DEX) by real-time PCR (Supplementary Fig S1A). The up to fourfold, GR-dependent induction of miR-379 in diabetic mice (Fig 1B) and its relatively high abundance in liver (Supplementary Fig S1B) next prompted us to investigate the general importance of this regulation in other models of elevated GC action. To this end, we analyzed liver extracts from healthy New Zealand black (NZB) mice compared to New Zealand obese (NZO) mice, the latter representing a multigenic obesity model (Leiter & Reifsnyder, 2004). In congruence with the hyperglucocorticoidemia in this model (Rose et al, 2010), miR-379 expression was found to be elevated significantly when compared to corresponding controls (Fig 1B), thereby correlating with circulating corticosterone levels in these mice (Supplementary Fig S1C), supporting the hypothesis that particularly miR-379 is a key output of the GC/GR endocrine pathway under conditions of metabolic dysfunction. In support of this possibility, levels of miR-379 were consistently found to be diminished in models of GR deficiency, including both miRNA-induced (Rose et al, 2011) and genetic (Opherk et al, 2004) hepatocyte-specific GR deficiency (Fig 1C and D, Supplementary Fig S1D). Indeed, chromatin immunoprecipitation using GR-specific antibody experimentally verified the recruitment of the receptor to a binding site within the miR-379 promoter (Fig 1E). A similar result had been previously reported in GR Chip-sequencing data deposited in human genome database (Supplementary Fig S1F), suggesting that the miR-379/410 cluster represents a direct transcriptional target of the GR. To verify the functional significance of GR DNA binding to the miR-379 locus in vivo, we examined mice harboring a dimerization-defective GR mutant (GRdim) that impairs receptor DNA binding to full GREs (Opherk et al, 2004). Wild-type mice chronically treated with DEX exhibited higher hepatic levels of miR-379 as compared with controls, while the genetic impairment of GR DNA-binding capacity in GRdim mice completely abrogated this effect (Supplementary Fig S1A), thereby validating a DNA binding-dependent regulatory function of the GC/GR pathway for miR-379 expression in the liver. Of note, DEX treatment of isolated primary mouse hepatocytes (Supplementary Fig S1E) and human liver slices (Fig 1F) led to the induction of miR-379 irrespective of culture glucose levels (data not shown), demonstrating the cell autonomy of the observed effects. Co-treatment with the GR-antagonist RU-486 completely abolished the DEX effects, thereby confirming GR specificity (Fig 1F). Figure 1. The miR-379/410 cluster is a downstream target of glucocorticoid signaling Heatmap showing relative miRNA expression between wild-type (wt) and db/db mice (n = 4), and between mice treated with negative control (NC) and GR-specific miRNA recombinant adeno-associated virus (rAAV) (n = 4). Higher and lower expression is displayed in red and green, respectively. Commonly regulated miRNAs in the miR-379/410 cluster are shown in bold. Differentially regulated miRNAs are ≥ twofold, P < 0.05. Quantitative miR-379 PCR levels in livers of db/db, New Zealand obese (NZO) mice, and corresponding controls—wt and New Zealand black (NZB) (n ≥ 4). Bar graphs show mean ± SEM; t-test: ***P < 0.001 or *P < 0.05. Quantitative miR-379 levels in livers of db/db mice treated with control or GR-specific shRNA adenovirus (n = 5). Bar graphs show mean ± SEM; t-test: *P < 0.05. Hepatic miR-379 levels as determined by RT–qPCR analysis in wt and hepatocyte-specific GR knockout mice (GR-AlfpCre) (n = 4). Bar graphs show mean ± SEM; t-test: ***P < 0.001. Chromatin immunoprecipitation (ChIP) qPCR for validation of GR-binding regions (GBR) upstream miR-379 hairpin: 1 (−11,197 to −111,268), 2 (−21,021 to −21,135), and 3 (−26,761 to −26,793). Fold enrichment of GR-binding site occupancy relative to negative control, anti-HA. (n = 4). Bar graphs show mean ± SEM; t-test: ***P < 0.001. miR-379 levels in human liver treated ex vivo with or without 1 μM RU486 and 0.1 μM DEX (n = 4). snoRNA-202 was used for normalization of miRNA levels. Bar graphs show mean ± SEM; ANOVA (with post hoc test): *P < 0.05. Download figure Download PowerPoint miR-379 silencing in liver affects circulating VLDL-TG levels The identification of miR-379 as a direct target of GC/GR signaling with aberrant hepatic expression in diabesity prompted us to next explore the functional importance of miR-379 for hepatic and systemic energy homeostasis. To this end, we efficiently and specifically silenced miR-379 in the liver by locked nucleic acid (LNA) antisense technology in wild-type animals (Fig 2A, Supplementary Fig S2A and C). While miR-379-specific LNA delivery had no influence on serum alanine aminotransferase (ALT) (Supplementary Fig S2B), hepatic glycogen (Supplementary Fig S2D), TG (Supplementary Fig S2E), and serum cholesterol (Supplementary Fig S2F) levels as well as glucose tolerance (Supplementary Fig S2G), miR-379 deficiency significantly lowered total serum TG levels (Fig 2B) and tended to decrease total serum cholesterol (Fig 2C), indicating that hepatic miR-379 principally controls metabolism of circulating TG. Indeed, serum fast-protein liquid chromatography (FPLC) revealed that liver silencing of miR-379 robustly reduced levels of VLDL-associated TG, while only mildly lowering cholesterol lipoprotein loading (Fig 2D, Supplementary Fig S2H). Consistent with the role of elevated VLDL-TG as a major independent risk factor for cardiovascular complications in the Metabolic Syndrome and its tight association with insulin resistance (Nordestgaard & Varbo, 2014), diminished VLDL-TG in miR-379 LNA-treated mice correlated with improved insulin sensitivity (Supplementary Fig S2I), inhibition of FOXO1 and phosphoenolpyruvate carboxykinase (PEPCK) (Supplementary Fig S2J and K), as well as consistent improvement in pyruvate tolerance (Supplementary Fig S2L), while blood glucose levels remained overall unaltered (Supplementary Fig S2M). Figure 2. miR-379 controls systemic VLDL triglyceride levels A. RT–qPCR analysis of miR-379 expression in C57Bl/6J (wt) mice treated with anti-miR-379 or scrambled control locked nucleic acid (LNA) (n = 5). Bar graphs show mean ± SEM; t-test: ***P < 0.001. B, C. Serum triglyceride (B) and cholesterol (C) levels after 1 and 2 weeks of LNA treatment of same animals as in (A) under fed conditions. Bar graphs show mean ± SEM; ANOVA (with post hoc test): **P < 0.01 or *P < 0.05. D. Triglyceride profiles of fast-protein liquid chromatography (FPLC)-fractionated serum of same animals as in (A). Very-low-density lipoprotein (VLDL) peak is indicated. E. Serum triglyceride levels of wt mice overexpressing miR-379 or scrambled control miRNA via hepatocyte-specific rAAV vectors (n = 4). Bar graphs show mean ± SEM; t-test: *P < 0.05. F. Triglyceride profiles of FPLC-fractionated serum of same animals as in (E). VLDL peak is indicated. Download figure Download PowerPoint To next determine whether elevation of hepatic miR-379 expression was sufficient to cause systemic dyslipidemia in a more chronic setting, we employed a recombinant adeno-associated virus (rAAV) delivery system allowing the expression of the miRNA specifically in liver parenchymal cells but not in other liver cell types for a period of several months (Rose et al, 2011). Eight weeks after rAAV delivery, mice with hepatocyte-specific miR-379 overexpression (Supplementary Fig S2N) that did not affect serum levels of liver damage markers (Supplementary Fig S2O) displayed significantly higher levels of serum TG associated with the VLDL fraction (Fig 2E and F) but maintained normal cholesterol homeostasis (Supplementary Fig S2P and Q). miR-379 acts through LSR- and LDLR-dependent hepatic lipid re-uptake The observed regulation of serum VLDL-TG by miR-379 next prompted us to explore the mechanistic basis of this effect. Systemic TG metabolism is determined by the relative balance of hepatic and peripheral lipid uptake and release, correlating with de novo TG formation (lipogenesis) and fatty acid (FA) β-oxidation in the liver (Wang et al, 2012). In this respect, miR-379-specific LNA treatment of mouse hepatocytes had no influence on FA oxidation as determined by metabolic flux analysis (Supplementary Fig S3A). Also, adipose tissue-associated lipoprotein lipase activity (Supplementary Fig S3B) and hepatic VLDL release were unaffected by miR-379 deficiency as compared to control littermates (Supplementary Fig S3C). In contrast, hepatic uptake of radiolabeled VLDL (Supplementary Fig S3D) and orally administered labeled TG (Fig 3A and B) was significantly and specifically elevated upon miR-379 knockdown in both db/db and wild-type mice, respectively, suggesting that the control of circulating VLDL-TG by hepatic miR-379 is not predominantly conferred by alterations in hepatic or peripheral lipid synthesis or utilization pathways but rather relies on enhanced hepatic VLDL-TG clearance. Figure 3. miR-379 controls systemic triglyceride levels via the lipolysis-stimulated lipoprotein receptor (LSR) and the low-density lipoprotein receptor (LDLR) A. Serum 3H-triolein in a lipid tolerance test in db/db mice treated with an anti-miR-379 or scrambled control Tough Decoy (TuD) construct delivered by rAAV (n = 5–6). Mice were fasted for 16 h and given a 100-μl oral fat load of olive oil spiked-in with 3H-triolein. Inset: area under the curve. Line graphs show mean ± SEM; ANOVA (with post hoc test): ***P < 0.001 or **P < 0.01. B. Organ distribution of 3H-triolein radioactivity from animals in (A). BAT, brown adipose tissue; pgWAT, perigonadal white adipose tissue; GCM, gastrocnemius skeletal muscle. Bar graphs show mean ± SEM; t-test: *P < 0.05. C. Protein levels of LSR and LDLR from livers of animals treated with anti-miR-379 or scrambled control LNA (n = 5), same animals as in Fig 2A. Shown are the LSR-α (68 kDa) and LSR-β (56 kDa) subunits and the glycosylated LDLR (between 100 and 130 kDa) protein. D. Protein levels of LSR and LDLR from livers of wild-type (wt) mice treated with dexamethasone (1 mg/kg BW) or isotonic saline for 28 days. (n = 7–8). E. Western blot of liver extracts from wt or LDLRKO mice treated with control or LSR shRNA-containing adenovirus and with anti-miR-379 or scrambled control LNA (n = 7). F. Serum triglyceride levels of same animals as in (E) under fed conditions. Bar graphs show mean ± SEM; ANOVA (with post hoc test): **P < 0.01 or *P < 0.05. G. Flag-LSR protein levels in HEK293 cells treated with miR-379 mimics. 100 nM cel-miR-293b was used as the control mimic. [-]: untransfected. H, I. miR-379 target validation of LSR (H) and LDLR (I) using a dual-luciferase reporter gene assay. miRNA binding site (MBS) predicted by RNA22 and MiRTiF and the corresponding mutated (mut) sequence were cloned into psiCHECK™-2 vector. Bar graphs show mean ± SEM; ANOVA (with post hoc test): ***P < 0.001. Download figure Download PowerPoint In congruence with this hypothesis, bioinformatic miRNA target gene screening (Supplementary Fig S4A) revealed that miR-379 was predicted to bind genes involved in hepatic lipid (re-) uptake, most notably including the major hepatic lipid re-uptake transporters, lipolysis-stimulated lipoprotein receptor (LSR, Supplementary Fig S3I), and low-density lipoprotein receptor (LDLR, Supplementary Fig S3J) (Ishibashi et al, 1993; Narvekar et al, 2009). Both, LSR and LDLR expression, have previously been found to be severely compromised in hypertriglyceridemic db/db mice, and liver-specific inactivation of both LSR and LDLR triggered the elevation of serum VLDL-TG in healthy wild-type animals by specifically preventing hepatic re-uptake of apolipoprotein B-associated TG from the circulation (Narvekar et al, 2009; Foley et al, 2013). In line with the in silico predictions, LNA-mediated miR-379 silencing led to the up-regulation of hepatic LSR and LDLR protein expression in wild-type animals (Fig 3C, Supplementary Fig S3K), and DEX treatment inhibited LSR and LDLR protein levels as compared with controls (Fig 3D, Supplementary Fig S3L), overall suggesting that miR-379 may regulate systemic VLDL-TG through its inhibitory impact on LSR and LDLR function. To address this hypothesis functionally, we performed genetic rescue experiments using LNA/shRNA adenovirus co-administration in wild-type mice, preventing the miR-379 LNA-dependent induction of LSR protein expression through simultaneous shRNA-mediated inhibition of LSR (Supplementary Fig S4B–D). Ten days after LNA/adenovirus co-delivery into the tail vein of mice, miR-379 silencing triggered hypotriglyceridemia (Supplementary Fig S4E) while LSR inhibition alone caused an elevation of circulating VLDL-TG as described when compared with control littermates (Narvekar et al, 2009). Of note, the treatments left cholesterol para
Referência(s)