Dynamic changes in DICER levels in adipose tissue control metabolic adaptations to exercise.

DICER is a key enzyme in microRNA (miRNA) biogenesis. Here we show that aerobic exercise training up-regulates DICER in adipose tissue of mice and humans. This can be mimicked by infusion of serum from exercised mice into sedentary mice and depends on AMPK-mediated signaling in both muscle and adipocytes. Adipocyte DICER is required for whole-body metabolic adaptations to aerobic exercise training, in part, by allowing controlled substrate utilization in adipose tissue, which, in turn, supports skeletal muscle function. Exercise training increases overall miRNA expression in adipose tissue, and up-regulation of miR-203-3p limits glycolysis in adipose under conditions of metabolic stress. We propose that exercise training-induced DICER-miR-203-3p up-regulation in adipocytes is a key adaptive response that coordinates signals from working muscle to promote whole-body metabolic adaptations.

s ·nr: a visual analytics framework for contextual analyses of private and public RNA-seq data.

BACKGROUND: Next-Generation Sequencing (NGS) has been widely accepted as an essential tool in molecular biology. Reduced costs and automated analysis pipelines make the use of NGS data feasible even for small labs, yet the methods for interpreting the data are not sophisticated enough to account for the amount of information. RESULTS: We propose s ·nr, a Visual Analytics tool that provides simple yet powerful visual interfaces for displaying and querying NGS data. It allows researchers to explore their own data in the context of experimental data deposited in public repositories, as well as to extract specific data sets with similar gene expression signatures. We tested s ·nr on 1543 RNA-Seq based mouse differential expression profiles derived from the public ArrayExpress platform. We provide the repository of processed data with this paper. CONCLUSION: s ·nr, easily deployable utilizing its containerized implementation, empowers researchers to analyze and relate their own RNA-Seq as well as to provide interactive and contextual crosstalk with data from public repositories. This allows users to deduce novel and unbiased hypotheses about the underlying molecular processes. DEMO: Login demo/demo: snr.sf.mpg.de (Tested with Google Chrome).

Obesity-induced overexpression of miR-802 impairs glucose metabolism through silencing of Hnf1b.

Insulin resistance represents a hallmark during the development of type 2 diabetes mellitus and in the pathogenesis of obesity-associated disturbances of glucose and lipid metabolism. MicroRNA (miRNA)-dependent post-transcriptional gene silencing has been recognized recently to control gene expression in disease development and progression, including that of insulin-resistant type 2 diabetes. The deregulation of miRNAs miR-143 (ref. 4), miR-181 (ref. 5), and miR-103 and miR-107 (ref. 6) alters hepatic insulin sensitivity. Here we report that the expression of miR-802 is increased in the liver of two obese mouse models and obese human subjects. Inducible transgenic overexpression of miR-802 in mice causes impaired glucose tolerance and attenuates insulin sensitivity, whereas reduction of miR-802 expression improves glucose tolerance and insulin action. We identify Hnf1b (also known as Tcf2) as a target of miR-802-dependent silencing, and show that short hairpin RNA (shRNA)-mediated reduction of Hnf1b in liver causes glucose intolerance, impairs insulin signalling and promotes hepatic gluconeogenesis. In turn, hepatic overexpression of Hnf1b improves insulin sensitivity in Lepr(db/db) mice. Thus, this study defines a critical role for deregulated expression of miR-802 in the development of obesity-associated impairment of glucose metabolism through targeting of Hnf1b, and assigns Hnf1b an unexpected role in the control of hepatic insulin sensitivity.

Decoding Lamarck-transgenerational control of metabolism by noncoding RNAs.

The concept of epigenetic transgenerational inheritance (ETI) posits that lifetime experiences in parents, particularly fathers, alter the phenotypic trajectory of their progeny independently of Mendelian genetics. Based on evidence from population studies and laboratory-controlled studies in syngenic animals, this long-term discredited so-called Lamarckian inheritance gained prominent attention. This article aims to summarize the current knowledge about ETI in lower and in higher organisms as well as in human cohorts and elaborates on epigenetic principles potentially underlying this nongenetic mode of heredity. Special attention is given to-small and long-noncoding RNAs in male gametes that recently emerged as a molecular sensor of organismal metabolic states which can ultimately relay information across the germline barrier by translating environmental cues into (epigenetic) changes in zygotic gene expression.

Cold-induced expression of a truncated adenylyl cyclase 3 acts as rheostat to brown fat function.

Promoting brown adipose tissue (BAT) activity innovatively targets obesity and metabolic disease. While thermogenic activation of BAT is well understood, the rheostatic regulation of BAT to avoid excessive energy dissipation remains ill-defined. Here, we demonstrate that adenylyl cyclase 3 (AC3) is key for BAT function. We identified a cold-inducible promoter that generates a 5' truncated AC3 mRNA isoform (Adcy3-at), whose expression is driven by a cold-induced, truncated isoform of PPARGC1A (PPARGC1A-AT). Male mice lacking Adcy3-at display increased energy expenditure and are resistant to obesity and ensuing metabolic imbalances. Mouse and human AC3-AT are retained in the endoplasmic reticulum, unable to translocate to the plasma membrane and lack enzymatic activity. AC3-AT interacts with AC3 and sequesters it in the endoplasmic reticulum, reducing the pool of adenylyl cyclases available for G-protein-mediated cAMP synthesis. Thus, AC3-AT acts as a cold-induced rheostat in BAT, limiting adverse consequences of cAMP activity during chronic BAT activation.

Growth-hormone-induced signal transducer and activator of transcription 5 signaling causes gigantism, inflammation, and premature death but protects mice from aggressive liver cancer.

UNLABELLED: Persistently high levels of growth hormone (GH) can cause liver cancer. GH activates multiple signal-transduction pathways, among them janus kinase (JAK) 2-signal transducer and activator of transcription (STAT) 5 (signal transducer and activator of transcription 5). Both hyperactivation and deletion of STAT5 in hepatocytes have been implicated in the development of hepatocellular carcinoma (HCC); nevertheless, the role of STAT5 in the development of HCC as a result of high GH levels remains enigmatic. Thus, we crossed a mouse model of gigantism and inflammatory liver cancer caused by hyperactivated GH signaling (GH(tg) ) to mice with hepatic deletion of STAT5 (STAT5(Δhep) ). Unlike GH(tg) mice, GH(tg) STAT5(Δhep) animals did not display gigantism. Moreover, the premature mortality, which was associated with chronic inflammation, as well as the pathologic alterations of hepatocytes observed in GH(tg) mice, were not observed in GH(tg) animals lacking STAT5. Strikingly, loss of hepatic STAT5 proteins led to enhanced HCC development in GH(tg) mice. Despite reduced chronic inflammation, GH(tg) STAT5(Δhep) mice displayed earlier and more advanced HCC than GH(tg) animals. This may be attributed to the combination of increased peripheral lipolysis, hepatic lipid synthesis, loss of hepatoprotective mediators accompanied by aberrant activation of tumor-promoting c-JUN and STAT3 signaling cascades, and accumulation of DNA damage secondary to loss of cell-cycle control. Thus, HCC was never observed in STAT5(Δhep) mice. CONCLUSION: As a result of their hepatoprotective functions, STAT5 proteins prevent progressive fatty liver disease and the formation of aggressive HCC in the setting of hyperactivated GH signaling. At the same time, they play a key role in controlling systemic inflammation and regulating organ and body size.

Nanopore sequencing unveils the complexity of the cold-activated murine brown adipose tissue transcriptome.

Alternative transcription increases transcriptome complexity by expression of multiple transcripts per gene. Annotation and quantification of transcripts using short-read sequencing is non-trivial. Long-read sequencing aims at overcoming these problems by sequencing full-length transcripts. Activation of brown adipose tissue (BAT) thermogenesis involves major transcriptomic remodeling and positively affects metabolism via increased energy expenditure. We benchmark Oxford Nanopore Technology (ONT) long-read sequencing protocols to Illumina short-read sequencing assessing alignment characteristics, gene and transcript detection and quantification, differential gene and transcript expression, transcriptome reannotation, and differential transcript usage (DTU). We find ONT sequencing is superior to Illumina for transcriptome reassembly, reducing the risk of false-positive events by unambiguously mapping reads to transcripts. We identified novel isoforms of genes undergoing DTU in cold-activated BAT including Cars2, Adtrp, Acsl5, Scp2, Aldoa, and Pde4d, validated by real-time PCR. The reannotated murine BAT transcriptome established here provides a framework for future investigations into the regulation of BAT.

Impairment of hepatic growth hormone and glucocorticoid receptor signaling causes steatosis and hepatocellular carcinoma in mice.

UNLABELLED: Growth hormone (GH)-activated signal transducer and activator of transcription 5 (STAT5) and the glucocorticoid (GC)-responsive glucocorticoid receptor (GR) are important signal integrators in the liver during metabolic and physiologic stress. Their deregulation has been implicated in the development of metabolic liver diseases, such as steatosis and progression to fibrosis. Using liver-specific STAT5 and GR knockout mice, we addressed their role in metabolism and liver cancer onset. STAT5 single and STAT5/GR double mutants developed steatosis, but only double-mutant mice progressed to liver cancer. Mechanistically, STAT5 deficiency led to the up-regulation of prolipogenic sterol regulatory element binding protein 1 (SREBP-1) and peroxisome proliferator activated receptor gamma (PPAR-γ) signaling. Combined loss of STAT5/GR resulted in GH resistance and hypercortisolism. The combination of both induced expression of adipose tissue lipases, adipose tissue lipid mobilization, and lipid flux to the liver, thereby aggravating STAT5-dependent steatosis. The metabolic dysfunctions in STAT5/GR compound knockout animals led to the development of hepatic dysplasia at 9 months of age. At 12 months, 35% of STAT5/GR-deficient livers harbored dysplastic nodules and ∼ 60% hepatocellular carcinomas (HCCs). HCC development was associated with GH and insulin resistance, enhanced tumor necrosis factor alpha (TNF-α) expression, high reactive oxygen species levels, and augmented liver and DNA damage parameters. Moreover, activation of the c-Jun N-terminal kinase 1 (JNK1) and STAT3 was prominent. CONCLUSION: Hepatic STAT5/GR signaling is crucial for the maintenance of systemic lipid homeostasis. Impairment of both signaling cascades causes severe metabolic liver disease and promotes spontaneous hepatic tumorigenesis.

Comprehensive Transcriptional Profiling and Mouse Phenotyping Reveals Dispensable Role for Adipose Tissue Selective Long Noncoding RNA Gm15551.

Cold and nutrient-activated brown adipose tissue (BAT) is capable of increasing systemic energy expenditure via the uncoupled respiration and secretion of endocrine factors, thereby protecting mice against diet-induced obesity and improving insulin response and glucose tolerance in men. Long non-coding RNAs (lncRNAs) have recently been identified as fine-tuning regulators of cellular function. While certain lncRNAs have been functionally characterised in adipose tissue, their overall contribution in the activation of BAT remains elusive. We identified lncRNAs correlating to interscapular brown adipose tissue (iBAT) function in a high fat diet (HFD) and cold stressed mice. We focused on Gm15551, which has an adipose tissue specific expression profile, is highly upregulated during adipogenesis, and downregulated by ß-adrenergic activation in mature adipocytes. Although we performed comprehensive transcriptional and adipocyte physiology profiling in vitro and in vivo, we could not detect an effect of gain or loss of function of Gm15551.

Signal transducer and activator of transcription 3 protects from liver injury and fibrosis in a mouse model of sclerosing cholangitis.

BACKGROUND & AIMS: Signal transducer and activator of transcription 3 (Stat3) is the main mediator of interleukin-6-type cytokine signaling required for hepatocyte proliferation and hepatoprotection, but its role in sclerosing cholangitis and other cholestatic liver diseases remains unresolved. METHODS: We investigated the role of Stat3 in inflammation-induced cholestatic liver injury and used mice lacking the multidrug resistance gene 2 (mdr2(-/-)) as a model for SC. RESULTS: We show that conditional inactivation of Stat3 in hepatocytes and cholangiocytes (stat3(Deltahc)) of mdr2(-/-) mice strongly aggravated bile acid-induced liver injury and fibrosis. A similar phenotype was observed in mdr2(-/-) mice lacking interleukin-6 production. Biochemical and molecular characterization suggested that Stat3 exerts hepatoprotective functions in both hepatocytes and cholangiocytes. Loss of Stat3 led to increased expression of tumor necrosis factor alpha, which might reduce the barrier function of bile ducts. Moreover, Stat3-deficient hepatocytes displayed up-regulation of bile acid biosynthesis genes and down-regulation of hepatoprotective epidermal growth factor receptor and insulin-like growth factor 1 signaling pathways. Consistently, stat3(Deltahc) mice were more sensitive to cholic acid-induced liver damage than control mice. CONCLUSIONS: Our data suggest that Stat3 prevents cholestasis and liver damage in sclerosing cholangitis via regulation of pivotal functions in hepatocytes and cholangiocytes.

Disruption of the growth hormone--signal transducer and activator of transcription 5--insulinlike growth factor 1 axis severely aggravates liver fibrosis in a mouse model of cholestasis.

UNLABELLED: Growth hormone (GH) resistance and low serum levels of insulinlike growth factor 1 (IGF-1) are common features in human liver fibrosis and cirrhosis. Signal transducer and activator of transcription 5 (STAT5) controls several vital functions in the liver, including GH-mediated transcription of IGF-1. To investigate the role of STAT5 in liver fibrogenesis, we specifically deleted the Stat5a/b locus both in hepatocytes and cholangiocytes in the multidrug resistance gene 2 knockout (Mdr2(-/-)) mouse model of cholestasis. Double knockout mice develop an early and severe liver fibrosis phenotype, accompanied by perturbed expression of key regulators of bile acid homeostasis. Deletion of Stat5 resulted in GH resistance, and IGF-1 levels in serum were undetectable. We could observe reduced expression of important hepatoprotective genes, such as epidermal growth factor receptor (Egfr), hepatocyte nuclear factor 6 (Hnf6), prolactin receptor (Prlr), and leukemia inhibitory factor receptor (Lifr) as well as increased numbers of apoptotic hepatocytes. CONCLUSION: Our data suggest that loss of STAT5 sensitizes hepatocytes to bile acid-induced damage and apoptosis caused by disruption of GH-induced transcription of Igf-1 and down-regulation of hepatoprotective genes. These findings could contribute to the understanding of liver fibrosis and future treatment strategies for liver fibrosis.

Salicylic diamines selectively eliminate residual undifferentiated cells from pluripotent stem cell-derived cardiomyocyte preparations.

Clinical translation of pluripotent stem cell (PSC) derivatives is hindered by the tumorigenic risk from residual undifferentiated cells. Here, we identified salicylic diamines as potent agents exhibiting toxicity to murine and human PSCs but not to cardiomyocytes (CMs) derived from them. Half maximal inhibitory concentrations (IC50) of small molecules SM2 and SM6 were, respectively, 9- and 18-fold higher for human than murine PSCs, while the IC50 of SM8 was comparable for both PSC groups. Treatment of murine embryoid bodies in suspension differentiation cultures with the most effective small molecule SM6 significantly reduced PSC and non-PSC contamination and enriched CM populations that would otherwise be eliminated in genetic selection approaches. All tested salicylic diamines exerted their toxicity by inhibiting the oxygen consumption rate (OCR) in PSCs. No or only minimal and reversible effects on OCR, sarcomeric integrity, DNA stability, apoptosis rate, ROS levels or beating frequency were observed in PSC-CMs, although effects on human PSC-CMs seemed to be more deleterious at higher SM-concentrations. Teratoma formation from SM6-treated murine PSC-CMs was abolished or delayed compared to untreated cells. We conclude that salicylic diamines represent promising compounds for PSC removal and enrichment of CMs without the need for other selection strategies.

Expression of activated STAT5 in neoplastic mast cells in systemic mastocytosis: subcellular distribution and role of the transforming oncoprotein KIT D816V.

Recent data suggest that the signal transducer and activator of transcription (STAT)5 contributes to differentiation and growth of mast cells. It has also been described that constitutively phosphorylated STAT5 (pSTAT5) plays a pro-oncogenic role in various myeloid neoplasms. We examined the expression of pSTAT5 in neoplastic mast cells in systemic mastocytosis and asked whether the disease-related oncoprotein KIT D816V is involved in STAT5 activation. As assessed by immunohistochemistry using the anti-pSTAT5 antibody AX1, neoplastic mast cells were found to display pSTAT5 in all SM patients examined (n = 40). Expression of pSTAT5 was also demonstrable in the KIT D816V-positive mast cell leukemia cell line HMC-1. Using various staining-protocols, pSTAT5 was found to be located in both the cytoplasmic and nuclear compartment of mast cells. To define the functional role of KIT D816V in STAT5-activation, Ba/F3 cells with doxycycline-inducible expression of KIT D816V were used. In these cells, induction of KIT D816V resulted in an increased expression of pSTAT5 without substantial increase in total STAT5. Moreover, the KIT D816V-targeting kinase-inhibitor PKC412 was found to counteract expression of pSTAT5 in HMC-1 cells as well as doxycycline-induced expression of pSTAT5 in Ba/F3 cells. Finally, a dominant negative STAT5-construct was found to inhibit growth of HMC-1 cells. Together, our data show that neoplastic mast cells express cytoplasmic and nuclear pSTAT5, that KIT D816V promotes STAT5-activation, and that STAT5-activation contributes to growth of neoplastic mast cells.

Senescence-Associated Metabolomic Phenotype in Primary and iPSC-Derived Mesenchymal Stromal Cells.

Long-term culture of primary cells is characterized by functional and secretory changes, which ultimately result in replicative senescence. It is largely unclear how the metabolome of cells changes during replicative senescence and if such changes are consistent across different cell types. We have directly compared culture expansion of primary mesenchymal stromal cells (MSCs) and induced pluripotent stem cell-derived MSCs (iMSCs) until they reached growth arrest. Both cell types acquired similar changes in morphology, in vitro differentiation potential, senescence-associated ß-galactosidase, and DNA methylation. Furthermore, MSCs and iMSCs revealed overlapping gene expression changes, particularly in functional categories related to metabolic processes. We subsequently compared the metabolomes of MSCs and iMSCs and observed overlapping senescence-associated changes in both cell types, including downregulation of nicotinamide ribonucleotide and upregulation of orotic acid. Taken together, replicative senescence is associated with a highly reproducible senescence-associated metabolomics phenotype, which may be used to monitor the state of cellular aging.

A MAFG-lncRNA axis links systemic nutrient abundance to hepatic glucose metabolism.

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease.

Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology.

In recent years, long noncoding RNAs (lncRNAs) have emerged as multifaceted regulators of gene expression, controlling key developmental and disease pathogenesis processes. However, due to the paucity of lncRNA loss-of-function mouse models, key questions regarding the involvement of lncRNAs in organism homeostasis and (patho)-physiology remain difficult to address experimentally in vivo. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 platform provides a powerful genome-editing tool and has been successfully applied across model organisms to facilitate targeted genetic mutations, including Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus. However, just a few lncRNA-deficient mouse lines have been created using CRISPR/Cas9-mediated genome engineering, presumably due to the need for lncRNA-specific gene targeting strategies considering the absence of open-reading frames in these loci. Here, we describe a step-wise procedure for the generation and validation of lncRNA loss-of-function mouse models using CRISPR/Cas9-mediated genome engineering. In a proof-of-principle approach, we generated mice deficient for the liver-enriched lncRNA Gm15441, which we found downregulated during development of metabolic disease and induced during the feeding/fasting transition. Further, we discuss guidelines for the selection of lncRNA targets and provide protocols for in vitro single guide RNA (sgRNA) validation, assessment of in vivo gene-targeting efficiency and knockout confirmation. The procedure from target selection to validation of lncRNA knockout mouse lines can be completed in 18⁻20 weeks, of which <10 days hands-on working time is required.

The different functions of Stat5 and chromatin alteration through Stat5 proteins.

Stat5 proteins modulate gene transcription upon cytokine- and growth factor action. Stat5a and Stat5b proteins alone are weak activators of transcription. They can modify chromatin organization through oligomerization and they act predominantly in co-operation and interaction with other proteins. The conservative view of exclusively nuclear functions of Stat5 was challenged by the observation of additional Stat5 effects in the cytoplasm, resulting in activation of the PI3K-Akt pathway. We summarize biological consequences of mutations in conserved domains of Stat5 or of deletions in the N- or C-terminal domains with impact on target gene transcription. Formation of higher-order oligomers is dramatically changed upon amino- or carboxyterminal deletions in Stat5 proteins. Mutations in or deletion of the Stat5 N-terminus leads to diminished leukemogenic potential of oncogenic Stat5, probably due to the inability to form Stat5 tetramers. The Stat5 N-terminal domain prevents persistent activation and can act as a DNA-docking platform for the glucocorticoid receptor (GR). The corresponding protocols should facilitate follow-up studies on Stat5 proteins and their contribution to normal physiological versus pathological processes through differential chromatin binding.

LincRNA H19 protects from dietary obesity by constraining expression of monoallelic genes in brown fat.

Increasing brown adipose tissue (BAT) thermogenesis in mice and humans improves metabolic health and understanding BAT function is of interest for novel approaches to counteract obesity. The role of long noncoding RNAs (lncRNAs) in these processes remains elusive. We observed maternally expressed, imprinted lncRNA H19 increased upon cold-activation and decreased in obesity in BAT. Inverse correlations of H19 with BMI were also observed in humans. H19 overexpression promoted, while silencing of H19 impaired adipogenesis, oxidative metabolism and mitochondrial respiration in brown but not white adipocytes. In vivo, H19 overexpression protected against DIO, improved insulin sensitivity and mitochondrial biogenesis, whereas fat H19 loss sensitized towards HFD weight gains. Strikingly, paternally expressed genes (PEG) were largely absent from BAT and we demonstrated that H19 recruits PEG-inactivating H19-MBD1 complexes and acts as BAT-selective PEG gatekeeper. This has implications for our understanding how monoallelic gene expression affects metabolism in rodents and, potentially, humans.

A microRNA screen reveals that elevated hepatic ectodysplasin A expression contributes to obesity-induced insulin resistance in skeletal muscle.

Over 40% of microRNAs (miRNAs) are located in introns of protein-coding genes, and many of these intronic miRNAs are co-regulated with their host genes. In such cases of co-regulation, the products of host genes and their intronic miRNAs can cooperate to coordinately regulate biologically important pathways. Therefore, we screened intronic miRNAs dysregulated in the livers of mouse models of obesity to identify previously uncharacterized protein-coding host genes that may contribute to the pathogenesis of obesity-associated insulin resistance and type 2 diabetes mellitus. Our approach revealed that expression of both the gene encoding ectodysplasin A (Eda), the causal gene in X-linked hypohidrotic ectodermal dysplasia (XLHED), and its intronic miRNA, miR-676, was increased in the livers of obese mice. Moreover, hepatic EDA expression is increased in obese human subjects and reduced upon weight loss, and its hepatic expression correlates with systemic insulin resistance. We also found that reducing miR-676 expression in db/db mice increases the expression of proteins involved in fatty acid oxidation and reduces the expression of inflammatory signaling components in the liver. Further, we found that Eda expression in mouse liver is controlled via PPARγ and RXR-α, increases in circulation under conditions of obesity, and promotes JNK activation and inhibitory serine phosphorylation of IRS1 in skeletal muscle. In accordance with these findings, gain- and loss-of-function approaches reveal that liver-derived EDA regulates systemic glucose metabolism, suggesting that EDA is a hepatokine that can contribute to impaired skeletal muscle insulin sensitivity in obesity.