scholarly journals RNA sequencing reveals niche gene expression effects of beta-hydroxybutyrate in primary myotubes

2021 ◽  
Vol 4 (10) ◽  
pp. e202101037
Author(s):  
Philip MM Ruppert ◽  
Lei Deng ◽  
Guido JEJ Hooiveld ◽  
Roland WJ Hangelbroek ◽  
Anja Zeigerer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Histone Deacetylase ◽  
Tca Cycle ◽  
Cell Types ◽  
Signaling Molecule ◽  
Histone Deacetylase Inhibition ◽  
Beneficial Effects ◽  
Beta Hydroxybutyrate

Various forms of fasting and ketogenic diet have shown promise in (pre-)clinical studies to normalize body weight, improve metabolic health, and protect against disease. Recent studies suggest that β-hydroxybutyrate (βOHB), a fasting-characteristic ketone body, potentially acts as a signaling molecule mediating its beneficial effects via histone deacetylase inhibition. Here, we have investigated whether βOHB, in comparison to the well-established histone deacetylase inhibitor butyrate, influences cellular differentiation and gene expression. In various cell lines and primary cell types, millimolar concentrations of βOHB did not alter differentiation in vitro, as determined by gene expression and histological assessment, whereas equimolar concentrations of butyrate consistently impaired differentiation. RNA sequencing revealed that unlike butyrate, βOHB minimally impacted gene expression in primary adipocytes, macrophages, and hepatocytes. However, in myocytes, βOHB up-regulated genes involved in the TCA cycle and oxidative phosphorylation, while down-regulating genes belonging to cytokine and chemokine signal transduction. Overall, our data do not support the notion that βOHB serves as a powerful signaling molecule regulating gene expression but suggest that βOHB may act as a niche signaling molecule in myocytes.

scholarly journals Transcriptomic analysis reveals niche gene expression effects of beta-hydroxybutyrate in primary myotubes

2021 ◽  
Author(s):  
Philip M. M. Ruppert ◽  
Guido J. E. J. Hooiveld ◽  
Roland W. J. Hangelbroek ◽  
Anja Zeigerer ◽  
Sander Kersten
Keyword(s):  
Gene Expression ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitor ◽  
Tca Cycle ◽  
Cell Types ◽  
C2c12 Myotubes ◽  
Signaling Molecule ◽  
Beneficial Effects ◽  
Deacetylase Inhibitor

ABSTRACTVarious forms of fasting, including time-restricted feeding, alternate day fasting, and periodic fasting have shown promise in clinical and pre-clinical studies to normalize body weight, improve metabolic health, and protect against disease. Recent studies suggest that β-hydroxybutyrate (βOHB), a characteristic ketone body of the fasted metabolic state, acts as a potential signaling molecule mediating the beneficial effects of the various forms of fasting, potentially by acting as a histone deacetylase inhibitor. In the first part we investigated whether βOHB, in comparison to the well-established histone deacetylase inhibitor butyrate, influences cellular differentiation in vitro. In C2C12 myotubes, 3T3-L1 adipocytes, and THP-1 monocytes, millimolar concentrations of βOHB did not alter differentiation, as determined by gene expression and histological assessment, whereas equimolar concentrations of butyrate potently impaired differentiation in all cell types. RNA-sequencing revealed that unlike butyrate, βOHB minimally impacted gene expression in adipocytes, macrophages, and hepatocytes. However, in myocytes, βOHB upregulated genes involved in the TCA cycle and oxidative phosphorylation, while downregulating genes belonging to cytokine and chemokine signal transduction. Overall, our data do not support the notion that βOHB serves as a powerful signaling molecule regulating gene expression in adipocytes, macrophages and hepatocytes, but suggest that βOHB may act as a niche signaling molecule in muscle.

scholarly journals Genome-Wide STAT3 Binding Analysis after Histone Deacetylase Inhibition Reveals Novel Target Genes in Dendritic Cells

2016 ◽  
Vol 9 (2) ◽  
pp. 126-144 ◽  
Author(s):  
Yaping Sun ◽  
Matthew Iyer ◽  
Richard McEachin ◽  
Meng Zhao ◽  
Yi-Mi Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dendritic Cells ◽  
Histone Deacetylase ◽  
Antigen Processing ◽  
Target Genes ◽  
Hdac Inhibition ◽  
Histone Deacetylase Inhibition ◽  
Immune Effector ◽  
Chip Sequencing ◽  
Genome Wide

STAT3 is a master transcriptional regulator that plays an important role in the induction of both immune activation and immune tolerance in dendritic cells (DCs). The transcriptional targets of STAT3 in promoting DC activation are becoming increasingly understood; however, the mechanisms underpinning its role in causing DC suppression remain largely unknown. To determine the functional gene targets of STAT3, we compared the genome-wide binding of STAT3 using ChIP sequencing coupled with gene expression microarrays to determine STAT3-dependent gene regulation in DCs after histone deacetylase (HDAC) inhibition. HDAC inhibition boosted the ability of STAT3 to bind to distinct DNA targets and regulate gene expression. Among the top 500 STAT3 binding sites, the frequency of canonical motifs was significantly higher than that of noncanonical motifs. Functional analysis revealed that after treatment with an HDAC inhibitor, the upregulated STAT3 target genes were those that were primarily the negative regulators of proinflammatory cytokines and those in the IL-10 signaling pathway. The downregulated STAT3-dependent targets were those involved in immune effector processes and antigen processing/presentation. The expression and functional relevance of these genes were validated. Specifically, functional studies confirmed that the upregulation of IL-10Ra by STAT3 contributed to the suppressive function of DCs following HDAC inhibition.

scholarly journals Enhancing droplet-based single-nucleus RNA-seq resolution using the semi-supervised machine learning classifier DIEM

2019 ◽  
Author(s):  
Marcus Alvarez ◽  
Elior Rahmani ◽  
Brandon Jew ◽  
Kristina M. Garske ◽  
Zong Miao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Types ◽  
Supervised Machine Learning ◽  
Data Sets ◽  
Rna Seq ◽  
Novel Approach ◽  
Single Nucleus ◽  
Downstream Analysis

AbstractSingle-nucleus RNA sequencing (snRNA-seq) measures gene expression in individual nuclei instead of cells, allowing for unbiased cell type characterization in solid tissues. Contrary to single-cell RNA seq (scRNA-seq), we observe that snRNA-seq is commonly subject to contamination by high amounts of extranuclear background RNA, which can lead to identification of spurious cell types in downstream clustering analyses if overlooked. We present a novel approach to remove debris-contaminated droplets in snRNA-seq experiments, called Debris Identification using Expectation Maximization (DIEM). Our likelihood-based approach models the gene expression distribution of debris and cell types, which are estimated using EM. We evaluated DIEM using three snRNA-seq data sets: 1) human differentiating preadipocytes in vitro, 2) fresh mouse brain tissue, and 3) human frozen adipose tissue (AT) from six individuals. All three data sets showed various degrees of extranuclear RNA contamination. We observed that existing methods fail to account for contaminated droplets and led to spurious cell types. When compared to filtering using these state of the art methods, DIEM better removed droplets containing high levels of extranuclear RNA and led to higher quality clusters. Although DIEM was designed for snRNA-seq data, we also successfully applied DIEM to single-cell data. To conclude, our novel method DIEM removes debris-contaminated droplets from single-cell-based data fast and effectively, leading to cleaner downstream analysis. Our code is freely available for use at https://github.com/marcalva/diem.

scholarly journals Single Cell, Single Nucleus and Spatial RNA Sequencing of the Human Liver Identifies Hepatic Stellate Cell and Cholangiocyte Heterogeneity

2021 ◽  
Author(s):  
Tallulah S Andrews ◽  
Jawairia Atif ◽  
Jeff C Liu ◽  
Catia T Perciani ◽  
Xue-Zhong Ma ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Human Liver ◽  
Stellate Cell ◽  
Parenchymal Cell ◽  
Cell Types ◽  
Cell Populations ◽  
Healthy Human ◽  
Single Nucleus

The critical functions of the human liver are coordinated through the interactions of hepatic parenchymal and non-parenchymal cells. Recent advances in single cell transcriptional approaches have enabled an examination of the human liver with unprecedented resolution. However, dissociation related cell perturbation can limit the ability to fully capture the human liver's parenchymal cell fraction, which limits the ability to comprehensively profile this organ. Here, we report the transcriptional landscape of 73,295 cells from the human liver using matched single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq). The addition of snRNA-seq enabled the characterization of interzonal hepatocytes at single-cell resolution, revealed the presence of rare subtypes of hepatic stellate cells previously only seen in disease, and detection of cholangiocyte progenitors that had only been observed during in vitro differentiation experiments. However, T and B lymphocytes and NK cells were only distinguishable using scRNA-seq, highlighting the importance of applying both technologies to obtain a complete map of tissue-resident cell-types. We validated the distinct spatial distribution of the hepatocyte, cholangiocyte and stellate cell populations by an independent spatial transcriptomics dataset and immunohistochemistry. Our study provides a systematic comparison of the transcriptomes captured by scRNA-seq and snRNA-seq and delivers a high-resolution map of the parenchymal cell populations in the healthy human liver.

scholarly journals Effectiveness of aronia berries for reduction of mild fibrosis and gene expression analysis in livers from mice fed a high-fat diet with aronia berries

2016 ◽  
Vol 6 (3) ◽  
pp. 144 ◽  
Author(s):  
Takuya Yamane ◽  
Miyuki Kozuka ◽  
Yoshio Yamamoto ◽  
Yoshihisa Nakano ◽  
Takenori Nakagaki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Liver Fibrosis ◽  
Beneficial Effect ◽  
Rna Sequencing ◽  
Expression Analysis ◽  
High Fat Diet ◽  
Gene Expression Analysis ◽  
High Fat ◽  
Expression Levels ◽  
Beneficial Effects

Background: Aronia berries have many potential effects on health, including an antioxidant effect, effect for antimutagenesis, hepatoprotection and cardioprotection, an antidiabetic effect and inhibition of cancer cell proliferation. Previous human studies have shown that aronia juice may be useful for treatment of obesity disorders.Objective: To reveal relationship between beneficial effect and the gene expression change by aronia berries, we analyzed mice livers using RNA sequencing and RT-qPCR.Method: At 28 days after starting a normal diet, a high fat diet and a high-fat diet containing 10% freeze-dried aronia berries, serum was obtained from veins of mice after isoflurane anesthesia, and liver tissues were isolated and weighed. Triglyceride, total cholesterol and LDL cholesterol levels were measured and total RNAs were extracted. cDNA libraries were prepared according to Illumina protocols and sequenced using an Illumina HiSeq2500 to perform 100 paired-end sequencing. RNA-sequence reads mapping was performed using a DNA nexus. Gene expression analysis was performed. The liver tissue specimens were fixed and embedded in paraffin. After 5-mm-thick paraffin sections had been cut, they were stained with hematoxylin-eosin using the standard procedure and also with Sirius Red.Results: In this study, we found that mild fibrosis induced by a high-fat diet was reduced in livers of mice fed a high-fat diet containing aronia berries. RNA sequencing and RT-qPCR analyses revealed that gene expression levels of Igfbp1 and Gadd45g were increased in livers from mice fed a high-fat diet containing aronia berries. Furthermore, results of an enzyme-linked immunoassay showed that a secreted protein levels of FABP1 and FABP4 were reduced in serum from mice fed a high-fat diet containing aronia berries. The results suggest that aronia berries have beneficial effects on mild fibrosis in liver.Conclusion: Aronia berries have a beneficial effect on liver fibrosis. The recovery from liver fibrosis is associated with expression levels of Gadd45g and Igfbp1 in the liver. The beneficial effects of aronia berries on liver fibrosis reduce the risk of liver cancer diseases and insulin resistance, resulting in reduction of serum FABP1 and FABP4 levels.Keywords: aronia; fibrosis; liver; Igfbp1; Gadd45g

scholarly journals Transcriptomic Modification in the Cerebral Cortex following Noninvasive Brain Stimulation: RNA-Sequencing Approach

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Ben Holmes ◽  
Seung Ho Jung ◽  
Jing Lu ◽  
Jessica A. Wagner ◽  
Liudmilla Rubbi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cerebral Cortex ◽  
Rna Sequencing ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Current Intensity ◽  
Beneficial Effects ◽  
Group A ◽  
The Impact

Transcranial direct current stimulation (tDCS) has been shown to modulate neuroplasticity. Beneficial effects are observed in patients with psychiatric disorders and enhancement of brain performance in healthy individuals has been observed following tDCS. However, few studies have attempted to elucidate the underlying molecular mechanisms of tDCS in the brain. This study was conducted to assess the impact of tDCS on gene expression within the rat cerebral cortex. Anodal tDCS was applied at 3 different intensities followed by RNA-sequencing and analysis. In each current intensity, approximately 1,000 genes demonstrated statistically significant differences compared to the sham group. A variety of functional pathways, biological processes, and molecular categories were found to be modified by tDCS. The impact of tDCS on gene expression was dependent on current intensity. Results show that inflammatory pathways, antidepressant-related pathways (GTP signaling, calcium ion binding, and transmembrane/signal peptide pathways), and receptor signaling pathways (serotonergic, adrenergic, GABAergic, dopaminergic, and glutamate) were most affected. Of the gene expression profiles induced by tDCS, some changes were observed across multiple current intensities while other changes were unique to a single stimulation intensity. This study demonstrates that tDCS can modify the expression profile of various genes in the cerebral cortex and that these tDCS-induced alterations are dependent on the current intensity applied.

Histone deacetylase inhibition is cytotoxic to oligodendrocyte precursor cells in vitro and in vivo

2016 ◽  
Vol 54 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Toros A. Dincman ◽  
Jason E. Beare ◽  
Sujata Saraswat Ohri ◽  
Vittorio Gallo ◽  
Michal Hetman ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Precursor Cells ◽  
Oligodendrocyte Precursor Cells ◽  
Histone Deacetylase Inhibition ◽  
Oligodendrocyte Precursor

scholarly journals Therapeutic Effect of Camel Milk and Its Exosomes on MCF7 Cells In Vitro and In Vivo

2018 ◽  
Vol 17 (4) ◽  
pp. 1235-1246 ◽  
Author(s):  
Abdelnaser A. Badawy ◽  
Mohammed A. El-Magd ◽  
Sana A. AlSadrah
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Immune Response ◽  
Local Injection ◽  
Camel Milk ◽  
Anticancer Effect ◽  
Mcf7 Cells ◽  
Beneficial Effects

Background/Objectives: In the Middle East, people consume camel milk regularly as it is believed to improve immunity against diseases and decrease the risk for cancer. Recently, it was noted that most of the beneficial effects of milk come from their nanoparticles, especially exosomes. Herein, we evaluated the anticancer potential of camel milk and its exosomes on MCF7 breast cancer cells (in vitro and in vivo) and investigated the possible underlying molecular mechanism of action. Methods/Results: Administration of camel milk (orally) and its exosomes (orally and by local injection) decreased breast tumor progression as evident by ( a) higher apoptosis (indicated by higher DNA fragmentation, caspase-3 activity, Bax gene expression, and lower Bcl2 gene expression), ( b) remarkable inhibition of oxidative stress (decrease in MDA levels and iNOS gene expression); ( c) induction of antioxidant status (increased activities of SOD, CAT, and GPX), ( d) notable reduction in expression of inflammation-( IL1b, NFκB), angiogenesis-( VEGF) and metastasis-( MMP9, ICAM1) related genes; and ( e) higher immune response (high number of CD+4, CD+8, NK1.1 T cells in spleen). Conclusions: Overall, administration of camel milk–derived exosomes showed better anticancer effect, but less immune response, than treatment by camel milk. Moreover, local injection of exosomes led to better improvement than oral administration. These findings suggest that camel milk and its exosomes have anticancer effect possibly through induction of apoptosis and inhibition of oxidative stress, inflammation, angiogenesis and metastasis in the tumor microenvironment. Thus, camel milk and its exosomes could be used as an anticancer agent for cancer treatment.

Abstract WP139: Intranasal Administration of Mesenchymal Stem Cell (MSC)-Derived Exosomes Confers Acute Neuroprotection After Neonatal Stroke

Stroke ◽  
2020 ◽  
Vol 51 (Suppl_1) ◽  
Author(s):  
Praneeti Pathipati ◽  
Joel Faustino ◽  
Matthieu Lecuyer ◽  
Jacqueline Strivelli ◽  
Donald Phinney ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Intranasal Administration ◽  
Therapeutic Option ◽  
Cell Types ◽  
Artery Occlusion ◽  
Neonatal Stroke ◽  
Beneficial Effects ◽  
The Status

Background: Brain injury caused by stroke is a surprisingly common occurrence in neonates and is associated with significant long-term disabilities. We and others have shown delayed mesenchymal stem cell (MSC)-based therapy to be beneficial after neonatal stroke. Mounting evidence suggests MSC-derived soluble factors as key mediators of their neuroprotective/regenerative effects. We wanted to test whether Exosomes (Exo) derived from MSC carry beneficial effects after neonatal stroke. Objectives: Characterize effects of intranasal administration of MSC-derived Exo after neonatal stroke. Methods: MSCs enriched from the bone marrow of C57Bl6 mice (immuno-depletion) were cultured for 3 days in Exo-free FBS and confirmed by flow cytometry to be CD44 + /CD29 + and CD11b - /CD45 - . Exo were isolated (ExoQuick, SBI), their size distribution determined (NanoSight™), and Exo labeled with CellVue® before intranasal administration. Postnatal day 9 (P9) mice were subjected to a 3h middle cerebral artery occlusion (tMCAO), Exo (5ug, 1uL in PBS) administered into the nostril ipsilateral to injury, and injury volume and cell types that uptake Exo determined. Results: By 24h after administration, labelled Exo were visible ipsilateral along the lateral ventricle, in the SVZ, corpus callosum and in the penumbra, localized largely to Glut1 + -vessels and Iba1 + -microglia (MG). By 72h, labeled Exo were predominantly localized in Iba1 + -MG peri-infarct. Very few Exo were seen contralateral. Compared to vehicle/untreated mice, intranasal Exo significantly reduced injury volume at 72h (p<0.01, n=5). Preliminary in vitro experiments using MG isolated from acutely injured neonatal brain (CD11b-conjugated beads) confirmed significantly higher Exo uptake by MG from the ipsilateral Vs. contralateral cortex (p<0.05, n=2). Summary: We demonstrate that MSC-Exo exert short-term protection against neonatal stroke and that the magnitude of Exo uptake depends on the status of MG activation after injury. We are characterizing longer-term effects of MSC-Exo on stroke outcome to further explore potential for intranasal MSC-Exo as a clinically suitable therapeutic option for neonatal stroke. Funding: CPA PG0816 (ZV); AHA Innovation Award 17IRG33430004 (ZV); R01HL139685 (ZV)

scholarly journals Sirtuin 3 Mediates Neuroprotection of Ketones against Ischemic Stroke

2015 ◽  
Vol 35 (11) ◽  
pp. 1783-1789 ◽  
Author(s):  
Junxiang Yin ◽  
Pengcheng Han ◽  
Zhiwei Tang ◽  
Qingwei Liu ◽  
Jiong Shi
Keyword(s):  
Ischemic Stroke ◽  
Ketone Bodies ◽  
Infarct Volume ◽  
Field Tests ◽  
Underlying Mechanism ◽  
Sirtuin 3 ◽  
Neuroprotective Effects ◽  
Beneficial Effects ◽  
Beta Hydroxybutyrate

Stroke is one of the leading causes of death. Growing evidence indicates that ketone bodies have beneficial effects in treating stroke, but their underlying mechanism remains unclear. Our previous study showed ketone bodies reduced reactive oxygen species by using NADH as an electron donor, thus increasing the NAD+/NADH ratio. In this study, we investigated whether mitochondrial NAD+-dependent Sirtuin 3 (SIRT3) could mediate the neuroprotective effects of ketone bodies after ischemic stroke. We injected mice with either normal saline or ketones (beta-hydroxybutyrate and acetoacetate) at 30 minutes after ischemia induced by transient middle cerebral artery (MCA) occlusion. We found that ketone treatment enhanced mitochondria function, reduced oxidative stress, and therefore reduced infarct volume. This led to improved neurologic function after ischemia, including the neurologic score and the performance in Rotarod and open field tests. We further showed that ketones' effects were achieved by upregulating NAD+-dependent SIRT3 and its downstream substrates forkhead box O3a (FoxO3a) and superoxide dismutase 2 (SOD2) in the penumbra region since knocking down SIRT3 in vitro diminished ketones' beneficial effects. These results provide us a foundation to develop novel therapeutics targeting this SIRT3-FoxO3a-SOD2 pathway.

Sign in / Sign up

Export Citation Format

Share Document