Blood-derived lysophospholipid sustains hepatic phospholipids and fat storage necessary for hepatoprotection in overnutrition.

The liver has a high demand for phosphatidylcholine (PC), particularly in overnutrition, where reduced phospholipid levels have been implicated in the development of nonalcoholic fatty liver disease (NAFLD). Whether other pathways exist in addition to de novo PC synthesis that contribute to hepatic PC pools remains unknown. Here, we identified the lysophosphatidylcholine (LPC) transporter major facilitator superfamily domain containing 2A (Mfsd2a) as critical for maintaining hepatic phospholipid pools. Hepatic Mfsd2a expression was induced in patients having NAFLD and in mice in response to dietary fat via glucocorticoid receptor action. Mfsd2a liver-specific deficiency in mice (L2aKO) led to a robust nonalcoholic steatohepatitis-like (NASH-like) phenotype within just 2 weeks of dietary fat challenge associated with reduced hepatic phospholipids containing linoleic acid. Reducing dietary choline intake in L2aKO mice exacerbated liver pathology and deficiency of liver phospholipids containing polyunsaturated fatty acids (PUFAs). Treating hepatocytes with LPCs containing oleate and linoleate, two abundant blood-derived LPCs, specifically induced lipid droplet biogenesis and contributed to phospholipid pools, while LPC containing the omega-3 fatty acid docosahexaenoic acid (DHA) promoted lipid droplet formation and suppressed lipogenesis. This study revealed that PUFA-containing LPCs drive hepatic lipid droplet formation, suppress lipogenesis, and sustain hepatic phospholipid pools - processes that are critical for protecting the liver from excess dietary fat.

The RNA-binding protein HuR is a negative regulator in adipogenesis.

Human antigen R (HuR) is an essential regulator of RNA metabolism, but its function in metabolism remains unclear. This study identifies HuR as a major repressor during adipogenesis. Knockdown and overexpression of HuR in primary adipocyte culture enhances and inhibits adipogenesis in vitro, respectively. Fat-specific knockout of HuR significantly enhances adipogenic gene program in adipose tissues, accompanied by a systemic glucose intolerance and insulin resistance. HuR knockout also results in depot-specific phenotypes: it can repress myogenesis program in brown fat, enhance inflammation program in epidydimal white fat and induce browning program in inguinal white fat. Mechanistically, HuR may inhibit adipogenesis by recognizing and modulating the stability of hundreds of adipocyte transcripts including Insig1, a negative regulator during adipogenesis. Taken together, our work establishes HuR as an important posttranscriptional regulator of adipogenesis and provides insights into how RNA processing contributes to adipocyte development.

cAMP-MicroRNA-203-IFNγ network regulates subcutaneous white fat browning and glucose tolerance.

OBJECTIVE: Brown and beige adipocytes in humans and rodents are specialized to burn lipids for heat generation as a natural defense against cold and obesity, which is advantageous to metabolic homeostasis. MicroRNAs as another regulatory layer to regulate metabolic homeostasis attracted a lot of attentions. Our previous work revealed microRNA (miR)-203 as a brown adipocyte-enriched microRNA involved in brown adipocytes development. However, the potential role of miR-203 in adipose tissue metabolic homeostasis has not been determined in vivo. In this study, we investigate the potential role of miR-203 in subcutaneous white adipose tissue (sub-WAT) browning and metabolic homeostasis. METHODS: We investigated the relationship between miR-203 and energy homeostasis in adipose tissue from cold exposed, high fat diet (HFD) fed, ob/ob and db/db mice. The functions of miR-203 on sub-WAT browning were validated through miR-203 knockdown or overexpression. The miR-203 targeted signal pathway was screened by RNAseq analysis. Luciferase report assay, western blot, and qPCR were performed to establish the miR-203 related upstream and downstream signal pathway in vivo and in vitro. The functions of miR-203 on obesity and metabolic homeostasis were validated through GTT/ITT and western blot on high fat diet-induced obesity in C57 mice. ELISA was used to determine the concentration of IFN-γ. Flow cytometry analysis was performed to determine the infiltration of macrophages in adipose tissue. RESULTS: MiR-203 expression positively correlates with energy expenditure, and overexpression of miR-203 could enhance sub-WAT browning in normal diet (ND) condition. Mechanistically, the expression of miR-203 is activated by cAMP-dependent C/EBPß up-regulation. Subsequently, miR-203 inhibits IFN-γ signal pathway activation by directly targeting Lyn, which is an activator of Jak1-Stat1. Moreover, the forced expression of miR-203 could improve insulin sensitivity and resist high fat diet-induced obesity by inhibiting IFN-γ. CONCLUSIONS: MicroRNA-203 (miR-203) promotes white adipose tissue browning in cold exposed mice and improves glucose tolerance in HFD fed mice by repressing IFN-γ. Since miR-203 is activated by cAMP-dependent C/EBPß up-regulation and directly represses IFN-γ signal pathway, we declare that miR-203 acts as a messenger between cAMP signal pathway and IFN-γ signal pathway.

Silencing an insulin-induced lncRNA, LncASIR, impairs the transcriptional response to insulin signalling in adipocytes.

Long noncoding RNA(lncRNA)s are new regulators governing the metabolism in adipose tissue. In this study, we aimed to understand how lncRNAs respond to insulin signalling and explore whether lncRNAs have a functional role in insulin signalling pathway. We treated primary adipocyte cultures with insulin and collected RNA for RNA-sequencing to profile the non-coding transcriptome changes, through which we identified a top Adipose Specific Insulin Responsive LncRNA (LncASIR). To determine its biological function, we knocked down LncASIR using dcas9-KRAB, followed by RNA-seq to examine the effect on insulin-induced gene expression program. We identified a set of lncRNAs regulated by insulin signalling pathway. LncASIR is transcribed from a super enhancer region and responds robustly to insulin treatment. Silencing LncASIR resulted in an impaired global insulin-responsive gene program. LncASIR is a novel and integral component in the insulin signalling pathway in adipocytes.

Identification of a natural beige adipose depot in mice.

Beige fat is a potential therapeutic target for obesity and other metabolic diseases due to its inducible brown fat-like functions. Inguinal white adipose tissue (iWAT) can undergo robust brown remodeling with appropriate stimuli and is therefore widely considered as a representative beige fat depot. However, adipose tissues residing in different anatomic depots exhibit a broad range of plasticity, raising the possibility that better beige fat depots with greater plasticity may exist. Here we identified and characterized a novel, naturally-existing beige fat depot, thigh adipose tissue (tAT). Unlike classic WATs, tAT maintains beige fat morphology at room temperature, whereas high-fat diet (HFD) feeding or aging promotes the development of typical WAT features, namely unilocular adipocytes. The brown adipocyte gene expression in tAT is consistently higher than in iWAT under cold exposure, HFD feeding, and rosiglitazone treatment conditions. Our molecular profiling by RNA-Seq revealed up-regulation of energy expenditure pathways and repressed inflammation in tAT relative to eWAT and iWAT. Furthermore, we demonstrated that the master fatty acid oxidation regulator peroxisome proliferator-activated receptor α is dispensable for maintaining and activating the beige character of tAT. Therefore, we have identified tAT as a natural beige adipose depot in mice with a unique molecular profile that does not require peroxisome proliferator-activated receptor α.

De novo reconstruction of human adipose transcriptome reveals conserved lncRNAs as regulators of brown adipogenesis.

Obesity has emerged as an alarming health crisis due to its association with metabolic risk factors such as diabetes, dyslipidemia, and hypertension. Recent work has demonstrated the multifaceted roles of lncRNAs in regulating mouse adipose development, but their implication in human adipocytes remains largely unknown. Here we present a catalog of 3149 adipose active lncRNAs, of which 909 are specifically detected in brown adipose tissue (BAT) by performing deep RNA-seq on adult subcutaneous, omental white adipose tissue and fetal BATs. A total of 169 conserved human lncRNAs show positive correlation with their nearby mRNAs, and knockdown assay supports a role of lncRNAs in regulating their nearby mRNAs. The knockdown of one of those, lnc-dPrdm16, impairs brown adipocyte differentiation in vitro and a significant reduction of BAT-selective markers in in vivo. Together, our work provides a comprehensive human adipose catalog built from diverse fat depots and establishes a roadmap to facilitate the discovery of functional lncRNAs in adipocyte development.

RNA Binding Protein Ybx2 Regulates RNA Stability During Cold-Induced Brown Fat Activation.

Recent years have seen an upsurge of interest in brown adipose tissue (BAT) to combat the epidemic of obesity and diabetes. How its development and activation are regulated at the posttranscriptional level, however, has yet to be fully understood. RNA binding proteins (RBPs) lie in the center of posttranscriptional regulation. To systemically study the role of RBPs in BAT, we profiled >400 RBPs in different adipose depots and identified Y-box binding protein 2 (Ybx2) as a novel regulator in BAT activation. Knockdown of Ybx2 blocks brown adipogenesis, whereas its overexpression promotes BAT marker expression in brown and white adipocytes. Ybx2-knockout mice could form BAT but failed to express a full thermogenic program. Integrative analysis of RNA sequencing and RNA-immunoprecipitation study revealed a set of Ybx2's mRNA targets, including Pgc1α, that were destabilized by Ybx2 depletion during cold-induced activation. Thus, Ybx2 is a novel regulator that controls BAT activation by regulating mRNA stability.

A complex association between DNA methylation and gene expression in human placenta at first and third trimesters.

The human placenta is a maternal-fetal organ essential for normal fetal development and maternal health. During pregnancy, the placenta undergoes many structural and functional changes in response to fetal needs and environmental exposures. Previous studies have demonstrated widespread epigenetic and gene expression changes from early to late pregnancy. However, on the global level, how DNA methylation changes impact on gene expression in human placenta is not yet well understood. We performed DNA methylome analysis by reduced representation bisulfite sequencing (RRBS) and gene expression analysis by RNA-Seq for both first and third trimester human placenta tissues. From first to third trimester, 199 promoters (corresponding to 189 genes) and 2,297 gene bodies were differentially methylated, with a clear dominance of hypermethylation (96.8% and 93.0% for promoters and gene bodies, respectively). A total of 2,447 genes were differentially expressed, of which 77.2% were down-regulated. Gene ontology analysis using differentially expressed genes were enriched for cell cycle and immune response functions. The correlation between DNA methylation and gene expression was non-linear and complex, depending on the genomic context (promoter or gene body) and gene expression levels. A wide range of DNA methylation and gene expression changes were observed at different gestational ages. The non-linear association between DNA methylation and gene expression indicates that epigenetic regulation of placenta development is more complex than previously envisioned.

Dynamic DNA methylation landscape defines brown and white cell specificity during adipogenesis.

OBJECTIVE: DNA methylation may be a stable epigenetic contributor to defining fat cell lineage. METHODS: We performed reduced representation bisulfite sequencing (RRBS) and RNA-seq to depict a genome-wide integrative view of the DNA methylome and transcriptome during brown and white adipogenesis. RESULTS: Our analysis demonstrated that DNA methylation is a stable epigenetic signature for brown and white cell lineage before, during, and after differentiation. We identified 31 genes whose promoters were significantly differentially methylated between white and brown adipogenesis at all three time points of differentiation. Among them, five genes belong to the Hox family; their expression levels were anti-correlated with promoter methylation, suggesting a regulatory role of DNA methylation in transcription. Blocking DNA methylation with 5-Aza-cytidine increased the expression of these genes, with the most prominent effect on Hoxc10, a repressor of BAT marker expression. CONCLUSIONS: Our data suggest that DNA methylation may play an important role in lineage-specific development in adipocytes.

Postnatal Deletion of Fat Storage-inducing Transmembrane Protein 2 (FIT2/FITM2) Causes Lethal Enteropathy.

Lipid droplets (LDs) are phylogenetically conserved cytoplasmic organelles that store neutral lipids within a phospholipid monolayer. LDs compartmentalize lipids and may help to prevent cellular damage caused by their excess or bioactive forms. FIT2 is a ubiquitously expressed transmembrane endoplasmic reticulum (ER) membrane protein that has previously been implicated in LD formation in mammalian cells and tissue. Recent data indicate that FIT2 plays an essential role in fat storage in an in vivo constitutive adipose FIT2 knock-out mouse model, but the physiological effects of postnatal whole body FIT2 depletion have never been studied. Here, we show that tamoxifen-induced FIT2 deletion using a whole body ROSA26CreER(T2)-driven FIT2 knock-out (iF2KO) mouse model leads to lethal intestinal pathology, including villus blunting and death of intestinal crypts, and loss of lipid absorption. iF2KO mice lose weight and die within 2 weeks after the first tamoxifen dose. At the cellular level, LDs failed to form in iF2KO enterocytes after acute oil challenge and instead accumulated within the ER. Intestinal bile acid transporters were transcriptionally dysregulated in iF2KO mice, leading to the buildup of bile acids within enterocytes. These data support the conclusion that FIT2 plays an essential role in regulating intestinal health and survival postnatally.

De Novo Reconstruction of Adipose Tissue Transcriptomes Reveals Long Non-coding RNA Regulators of Brown Adipocyte Development.

Brown adipose tissue (BAT) protects against obesity by promoting energy expenditure via uncoupled respiration. To uncover BAT-specific long non-coding RNAs (lncRNAs), we used RNA-seq to reconstruct de novo transcriptomes of mouse brown, inguinal white, and epididymal white fat and identified ∼1,500 lncRNAs, including 127 BAT-restricted loci induced during differentiation and often targeted by key regulators PPARγ, C/EBPα, and C/EBPß. One of them, lnc-BATE1, is required for establishment and maintenance of BAT identity and thermogenic capacity. lnc-BATE1 inhibition impairs concurrent activation of brown fat and repression of white fat genes and is partially rescued by exogenous lnc-BATE1 with mutated siRNA-targeting sites, demonstrating a function in trans. We show that lnc-BATE1 binds heterogeneous nuclear ribonucleoprotein U and that both are required for brown adipogenesis. Our work provides an annotated catalog for the study of fat depot-selective lncRNAs and establishes lnc-BATE1 as a regulator of BAT development and physiology.

Improved reduced representation bisulfite sequencing for epigenomic profiling of clinical samples.

BACKGROUND: DNA methylation plays crucial roles in epigenetic gene regulation in normal development and disease pathogenesis. Efficient and accurate quantification of DNA methylation at single base resolution can greatly advance the knowledge of disease mechanisms and be used to identify potential biomarkers. We developed an improved pipeline based on reduced representation bisulfite sequencing (RRBS) for cost-effective genome-wide quantification of DNA methylation at single base resolution. A selection of two restriction enzymes (TaqαI and MspI) enables a more unbiased coverage of genomic regions of different CpG densities. We further developed a highly automated software package to analyze bisulfite sequencing results from the Solexa GAIIx system. RESULTS: With two sequencing lanes, we were able to quantify ~1.8 million individual CpG sites at a minimum sequencing depth of 10. Overall, about 76.7% of CpG islands, 54.9% of CpG island shores and 52.2% of core promoters in the human genome were covered with at least 3 CpG sites per region. CONCLUSIONS: With this new pipeline, it is now possible to perform whole-genome DNA methylation analysis at single base resolution for a large number of samples for understanding how DNA methylation and its changes are involved in development, differentiation, and disease pathogenesis.

DNMT1 and AIM1 Imprinting in human placenta revealed through a genome-wide screen for allele-specific DNA methylation.

BACKGROUND: Genomic imprinting is an epigenetically regulated process wherein genes are expressed in a parent-of-origin specific manner. Many imprinted genes were initially identified in mice; some of these were subsequently shown not to be imprinted in humans. Such discrepancy reflects developmental, morphological and physiological differences between mouse and human tissues. This is particularly relevant for the placenta. Study of genomic imprinting thus needs to be carried out in a species and developmental stage-specific manner. We describe here a new strategy to study allele-specific DNA methylation in the human placenta for the discovery of novel imprinted genes. RESULTS: Using this methodology, we confirmed 16 differentially methylated regions (DMRs) associated with known imprinted genes. We chose 28 genomic regions for further testing and identified two imprinted genes (DNMT1 and AIM1). Both genes showed maternal allele-specific methylation and paternal allele-specific transcription. Imprinted expression for AIM1 was conserved in the cynomolgus macaque placenta, but not in other macaque tissues or in the mouse. CONCLUSIONS: Our study indicates that while there are many genomic regions with allele-specific methylation in tissues like the placenta, only a small sub-set of them are associated with allele-specific transcription, suggesting alternative functions for such genomic regions. Nonetheless, novel tissue-specific imprinted genes remain to be discovered in humans. Their identification may help us better understand embryonic and fetal development.

Tet1 regulates adult hippocampal neurogenesis and cognition.

DNA hydroxylation catalyzed by Tet dioxygenases occurs abundantly in embryonic stem cells and neurons in mammals. However, its biological function in vivo is largely unknown. Here, we demonstrate that Tet1 plays an important role in regulating neural progenitor cell proliferation in adult mouse brain. Mice lacking Tet1 exhibit impaired hippocampal neurogenesis accompanied by poor learning and memory. In adult neural progenitor cells deficient in Tet1, a cohort of genes involved in progenitor proliferation were hypermethylated and downregulated. Our results indicate that Tet1 is positively involved in the epigenetic regulation of neural progenitor cell proliferation in the adult brain.

Global DNA hypermethylation in down syndrome placenta.

Down syndrome (DS), commonly caused by an extra copy of chromosome 21 (chr21), occurs in approximately one out of 700 live births. Precisely how an extra chr21 causes over 80 clinically defined phenotypes is not yet clear. Reduced representation bisulfite sequencing (RRBS) analysis at single base resolution revealed DNA hypermethylation in all autosomes in DS samples. We hypothesize that such global hypermethylation may be mediated by down-regulation of TET family genes involved in DNA demethylation, and down-regulation of REST/NRSF involved in transcriptional and epigenetic regulation. Genes located on chr21 were up-regulated by an average of 53% in DS compared to normal villi, while genes with promoter hypermethylation were modestly down-regulated. DNA methylation perturbation was conserved in DS placenta villi and in adult DS peripheral blood leukocytes, and enriched for genes known to be causally associated with DS phenotypes. Our data suggest that global epigenetic changes may occur early in development and contribute to DS phenotypes.

Measuring the methylome in clinical samples: improved processing of the Infinium Human Methylation450 BeadChip Array.

The Infinium Human Methylation450 BeadChip Array (TM) (Infinium 450K) is an important tool for studying epigenetic patterns associated with disease. This array offers a high-throughput, low cost alternative to more comprehensive sequencing-based methodologies. Here we compare data generated by interrogation of the same seven clinical samples by Infinium 450K and reduced representation bisulfite sequencing (RRBS). This is the largest data set comparing Infinium 450K array to the comprehensive RRBS methodology reported so far. We show good agreement between the two methodologies. A read depth of four or more reads in the RRBS data was sufficient to achieve good agreement with Infinium 450K. However, we observe that intermediate methylation values (20-80%) are more variable between technologies than values at the extremes of the bimodal methylation distribution. We describe careful processing of Infinium 450K data to correct for known limitations and batch effects. Using methodologies proposed by others and newly implemented and combined in this report, agreement of Infinium 450K data with independent techniques can be vastly improved.

Mixed learning algorithms and features ensemble in hepatotoxicity prediction.

Drug-induced liver injury, although infrequent, is an important safety concern that can lead to fatality in patients and failure in drug developments. In this study, we have used an ensemble of mixed learning algorithms and mixed features for the development of a model to predict hepatic effects. This robust method is based on the premise that no single learning algorithm is optimum for all modelling problems. An ensemble model of 617 base classifiers was built from a diverse set of 1,087 compounds. The ensemble model was validated internally with five-fold cross-validation and 25 rounds of y-randomization. In the external validation of 120 compounds, the ensemble model had achieved an accuracy of 75.0%, sensitivity of 81.9% and specificity of 64.6%. The model was also able to identify 22 of 23 withdrawn drugs or drugs with black box warning against hepatotoxicity. Dronedarone which is associated with severe liver injuries, announced in a recent FDA drug safety communication, was predicted as hepatotoxic by the ensemble model. It was found that the ensemble model was capable of classifying positive compounds (with hepatic effects) well, but less so on negatives compounds when they were structurally similar. The ensemble model built in this study is made available for public use.