Transcriptomic and Metabolomic Studies Reveal That Toll-like Receptor 2 Has a Role in Glucose-Related Metabolism in Unchallenged Zebrafish Larvae (Danio rerio)

Simple Summary Toll-like receptor 2 (TLR2) has been demonstrated to participate in the progression of some metabolic disorders due to its role as a pro-inflammatory trigger. However, whether TLR2 plays a role in mediating metabolism under an unchallenged condition is still unknown. Therefore, we utilized zebrafish larvae as an in vivo model to investigate the metabolic control functions of TLR2 through transcriptomic and metabolomic approaches at a whole-organism level. We found that the concentration of glucose, lactate, succinate, and malate is higher in a tlr2 mutant which is associated with lower expression of genes involved in the glycolysis and gluconeogenesis pathways. These results demonstrate that tlr2 plays a role in controlling glucose metabolism homeostasis. Abstract Toll-like receptors (TLRs) have been implicated in the regulation of various metabolism pathways, in addition to their function in innate immunity. Here, we investigate the metabolic function of TLR2 in a larval zebrafish system. We studied larvae from a tlr2 mutant and the wild type sibling controls in an unchallenged normal developmental condition using transcriptomic and metabolomic analyses methods. RNAseq was used to evaluate transcriptomic differences between the tlr2 mutant and wild-type control zebrafish larvae and found a signature set of 149 genes to be significantly altered in gene expression. The expression level of several genes was confirmed by qPCR analyses. Gene set enrichment analysis (GSEA) revealed differential enrichment of genes between the two genotypes related to valine, leucine, and isoleucine degradation and glycolysis and gluconeogenesis. Using 1H nuclear magnetic resonance (NMR) metabolomics, we found that glucose and various metabolites related with glucose metabolism were present at higher levels in the tlr2 mutant. Furthermore, we confirmed that the glucose level is higher in tlr2 mutants by using a fluorometric assay. Therefore, we have shown that TLR2, in addition to its function in immunity, has a function in controlling metabolism during vertebrate development. The functions are associated with transcriptional regulation of various enzymes involved in glucose metabolism that could explain the different levels of glucose, lactate, succinate, and malate in larvae of a tlr2 mutant.

[1]  H. Spaink,et al.  Specificity of the innate immune responses to different classes of non-tuberculous mycobacteria , 2023, Frontiers in Immunology.

[2]  A. Meijer,et al.  Repurposing Tamoxifen as Potential Host-Directed Therapeutic for Tuberculosis , 2022, mBio.

[3]  R. Martindale,et al.  Gut Microbiome and Its Impact on Obesity and Obesity-Related Disorders , 2022, Current Gastroenterology Reports.

[4]  Daijie Chen,et al.  Disturbances of the Gut Microbiota and Microbiota-Derived Metabolites in Inflammatory Bowel Disease , 2022, Nutrients.

[5]  Ling Yang,et al.  The critical role of gut microbiota in obesity , 2022, Frontiers in Endocrinology.

[6]  S. K. Saikia,et al.  Use of Zebrafish as a Model Organism to Study Oxidative Stress: A Review. , 2022, Zebrafish.

[7]  H. Spaink,et al.  Leptin mutation and mycobacterial infection lead non-synergistically to a similar metabolic syndrome , 2022, Metabolomics.

[8]  M. Norazmi,et al.  Immunometabolism of Immune Cells in Mucosal Environment Drives Effector Responses against Mycobacterium tuberculosis , 2022, International journal of molecular sciences.

[9]  L. Ramakrishnan,et al.  mTOR-associated Mitochondrial Energy Metabolism Limits Mycobacterium ESX-1-induced Cytotoxicity , 2022, The Journal of Immunology.

[10]  H. Spaink,et al.  The Role of TLR2 in Infectious Diseases Caused by Mycobacteria: From Cell Biology to Therapeutic Target , 2022, Biology.

[11]  E. Pearce,et al.  mTOR-regulated mitochondrial metabolism limits mycobacterium-induced cytotoxicity , 2022, Cell.

[12]  R. Zhao,et al.  A Novel Function of Mitochondrial Phosphoenolpyruvate Carboxykinase as a Regulator of Inflammatory Response in Kupffer Cells , 2021, Frontiers in Cell and Developmental Biology.

[13]  T. Hankemeier,et al.  Metabolomic and transcriptomic profiling of adult mice and larval zebrafish leptin mutants reveal a common pattern of changes in metabolites and signaling pathways , 2021, Cell & Bioscience.

[14]  Roeland M. H. Merks,et al.  A Novel Function of TLR2 and MyD88 in the Regulation of Leukocyte Cell Migration Behavior During Wounding in Zebrafish Larvae , 2021, Frontiers in Cell and Developmental Biology.

[15]  S. Mandal,et al.  LncRNA HOTAIR regulates glucose transporter Glut1 expression and glucose uptake in macrophages during inflammation , 2021, Scientific reports.

[16]  J. M. Brown,et al.  The Gut Microbial Endocrine Organ in Type 2 Diabetes , 2020, Endocrinology.

[17]  L. Conti,et al.  Toll-Like Receptor 2 at the Crossroad between Cancer Cells, the Immune System, and the Microbiota , 2020, International journal of molecular sciences.

[18]  S. A. Patten,et al.  A Great Catch for Investigating Inborn Errors of Metabolism—Insights Obtained from Zebrafish , 2020, Biomolecules.

[19]  W. Petri,et al.  TLR2 as a Therapeutic Target in Bacterial Infection. , 2020, Trends in molecular medicine.

[20]  R. Zhou,et al.  DAMP-sensing receptors in sterile inflammation and inflammatory diseases , 2019, Nature Reviews Immunology.

[21]  R. Marín-Juez,et al.  Infection and RNA-seq analysis of a zebrafish tlr2 mutant shows a broad function of this toll-like receptor in transcriptional and metabolic control and defense to Mycobacterium marinum infection , 2019, bioRxiv.

[22]  A. Meijer,et al.  Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages , 2019, bioRxiv.

[23]  H. Spaink,et al.  Intestinal microbiome adjusts the innate immune setpoint during colonization through negative regulation of MyD88 , 2018, Nature Communications.

[24]  Kyongbum Lee,et al.  Gut Microbiota-Derived Tryptophan Metabolites Modulate Inflammatory Response in Hepatocytes and Macrophages , 2018, Cell reports.

[25]  Joseph G Ibrahim,et al.  Heavy-tailed prior distributions for sequence count data: removing the noise and preserving large differences , 2018, bioRxiv.

[26]  J. Schiller,et al.  Metabolic profiling of zebrafish (Danio rerio) embryos by NMR spectroscopy reveals multifaceted toxicity of β-methylamino-L-alanine (BMAA) , 2017, Scientific Reports.

[27]  Rob Patro,et al.  Salmon provides fast and bias-aware quantification of transcript expression , 2017, Nature Methods.

[28]  J. Berry,et al.  High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance of Intact Zebrafish Embryos Detects Metabolic Changes Following Exposure to Teratogenic Polymethoxyalkenes from Algae. , 2016, Zebrafish.

[29]  L. Joosten,et al.  Rewiring cellular metabolism via the AKT/mTOR pathway contributes to host defence against Mycobacterium tuberculosis in human and murine cells , 2016, European journal of immunology.

[30]  Jensen H C Yiu,et al.  Interaction between gut microbiota and toll-like receptor: from immunity to metabolism , 2016, Journal of Molecular Medicine.

[31]  S. Han,et al.  Lipoteichoic acids as a major virulence factor causing inflammatory responses via Toll-like receptor 2 , 2016, Archives of pharmacal research.

[32]  Matthew Stephens,et al.  False discovery rates: a new deal , 2016, bioRxiv.

[33]  R. Marín-Juez,et al.  Common and specific downstream signaling targets controlled by Tlr2 and Tlr5 innate immune signaling in zebrafish , 2015, BMC Genomics.

[34]  Gang Liu,et al.  The Monocarboxylate Transporter 4 Is Required for Glycolytic Reprogramming and Inflammatory Response in Macrophages* , 2014, The Journal of Biological Chemistry.

[35]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[36]  Wouter J. Veneman,et al.  Comparative studies of Toll-like receptor signalling using zebrafish. , 2014, Developmental and comparative immunology.

[37]  F. Viñals,et al.  Mitochondrial Phosphoenolpyruvate Carboxykinase (PEPCK-M) Is a Pro-survival, Endoplasmic Reticulum (ER) Stress Response Gene Involved in Tumor Cell Adaptation to Nutrient Availability* , 2014, The Journal of Biological Chemistry.

[38]  Wouter J. Veneman,et al.  Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery , 2014, Journal of visualized experiments : JoVE.

[39]  Kathryn E. Crosier,et al.  Epidermal cells help coordinate leukocyte migration during inflammation through fatty acid-fuelled matrix metalloproteinase production , 2014, Nature Communications.

[40]  A. Harris,et al.  PCK2 activation mediates an adaptive response to glucose depletion in lung cancer , 2014, Oncogene.

[41]  Maxim N. Artyomov,et al.  TLR-driven early glycolytic reprogramming via the kinases TBK1-IKKɛ supports the anabolic demands of dendritic cell activation , 2014, Nature Immunology.

[42]  Chih-Hao Chang,et al.  Fueling Immunity: Insights into Metabolism and Lymphocyte Function , 2013, Science.

[43]  Annemarie H Meijer,et al.  Robotic injection of zebrafish embryos for high-throughput screening in disease models. , 2013, Methods.

[44]  B. Faubert,et al.  Posttranscriptional Control of T Cell Effector Function by Aerobic Glycolysis , 2013, Cell.

[45]  E. Pearce,et al.  Metabolic pathways in immune cell activation and quiescence. , 2013, Immunity.

[46]  Wouter J. Veneman,et al.  A zebrafish high throughput screening system used for Staphylococcus epidermidis infection marker discovery , 2013, BMC Genomics.

[47]  P. Massari,et al.  The Role of TLR2 in Infection and Immunity , 2012, Front. Immun..

[48]  R. Deberardinis,et al.  Cellular Metabolism and Disease: What Do Metabolic Outliers Teach Us? , 2012, Cell.

[49]  A. Ordas,et al.  Comparison of static immersion and intravenous injection systems for exposure of zebrafish embryos to the natural pathogen Edwardsiella tarda , 2011, BMC Immunology.

[50]  C. Dang,et al.  Otto Warburg's contributions to current concepts of cancer metabolism , 2011, Nature Reviews Cancer.

[51]  Chi V. Dang,et al.  Otto Warburg's contributions to current concepts of cancer metabolism , 2011, Nature Reviews Cancer.

[52]  O. Soehnlein,et al.  Phagocyte partnership during the onset and resolution of inflammation , 2010, Nature Reviews Immunology.

[53]  H. Spaink,et al.  In vivo metabolite profile of adult zebrafish brain obtained by high‐resolution localized magnetic resonance spectroscopy , 2009, Journal of magnetic resonance imaging : JMRI.

[54]  E. Beale,et al.  PCK1 and PCK2 as candidate diabetes and obesity genes , 2007, Cell Biochemistry and Biophysics.

[55]  Pablo Tamayo,et al.  Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[56]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[57]  K. Jungermann,et al.  Human mitochondrial phosphoenolpyruvate carboxykinase 2 gene. Structure, chromosomal localization and tissue-specific expression. , 1998, The Biochemical journal.