bims-aditis Biomed News
on Adipose tissue, inflammation, immunometabolism
Issue of 2022–02–20
seventeen papers selected by
Matthew C. Sinton, University of Glasgow
  1. A Clearing Method for Three-Dimensional Imaging of Adipose Tissue.
  2. Fluorescent Genetic Tools for Studying Brown Fat Development and Function in Mice.
  3. Metabolic Turnover Studies to Quantify Energy Uptake by Thermogenic Adipose Tissues of Mice.
  4. Activation of UCP1-Independent Ca2+ Cycling Thermogenesis by Wireless Optogenetics.
  5. Adipose mitochondrial complex I deficiency modulates inflammation and glucose homeostasis in a sex-dependent manner.
  6. Impaired phosphocreatine metabolism in white adipocytes promotes inflammation.
  7. Analysis of Single-Cell/Nucleus Transcriptome Data in Adipose Tissue.
  8. Isolation and Characterization of Human Brown Adipocytes.
  9. Inhibition of the Monocarboxylate Transporter 1 (MCT1) Promotes 3T3-L1 Adipocyte Proliferation and Enhances Insulin Sensitivity.
  10. γδ T cells monitor tissue health.
  11. Modulation of Macrophage Immunometabolism: A New Approach to Fight Infections.
  12. Brown Fat Anatomy in Humans and Rodents.
  13. Activating Human Adipose Tissue with the β3-Adrenergic Agonist Mirabegron.
  14. Zinc Levels Affect the Metabolic Switch of T Cells by Modulating Glucose Uptake and Insulin Receptor Signaling.
  15. Sleep Disturbance and Metabolic Dysfunction: The Roles of Adipokines.
  16. IL-21 plays an important role in modulating "Th17-Treg" cell axis in leprosy Type 1 reactions.
  17. Stable Isotope Tracing and Metabolomics to Study In Vivo Brown Adipose Tissue Metabolic Fluxes.
  1. Methods Mol Biol. 2022 ;2448 73-82
    A Clearing Method for Three-Dimensional Imaging of Adipose Tissue.
    Zeran Lin, Jingyi Chi, Paul Cohen.
      Adipose tissue is not simply a collection of lipid storing fat cells, but is now recognized to be a complex tissue with a central role in whole body energy homeostasis. In order to understand how adipocytes and associated cell types interact in normal physiology and in pathological states like obesity, it is critical to obtain a holistic view of cells and structures in three dimensions. To that end, we have adapted the iDISCO/iDISCO+ tissue clearing protocol to facilitate the delipidation of fat tissues, while still maintaining their architecture. We describe here this method, that we refer to as Adipo-Clear, highlighting key steps in the protocol as well as important technical considerations. This versatile approach can provide entirely new insights into adipose tissue biology in health and disease.
    Keywords:  3D imaging; Adipose tissue; Light-sheet fluorescence microscopy; Tissue clearing; Whole mount immunostaining
    DOI:  https://doi.org/10.1007/978-1-0716-2087-8_4
  2. Methods Mol Biol. 2022 ;2448 203-215
    Fluorescent Genetic Tools for Studying Brown Fat Development and Function in Mice.
    Rajib Mukherjee, Joan Sanchez-Gurmaches.
      Techniques to trace and isolate brown adipocyte precursor and adipocytes during development and disease are essential to fully understand brown adipose tissue development and function. Here we report several protocols using the R26R-mTmG reporter mice in thermogenic tissues based on confocal microscopy and fluorescence based flow cytometry. These techniques may be useful to understand the influence of genetic or environmental alterations in brown adipocyte precursors and adipocyte biology.
    Keywords:  Adipocyte precursor cells; Clearing; Confocal microscopy; Development; Flow cytometry; Immunofluorescence; Lineage tracing; mTmG
    DOI:  https://doi.org/10.1007/978-1-0716-2087-8_13
  3. Methods Mol Biol. 2022 ;2448 107-118
    Metabolic Turnover Studies to Quantify Energy Uptake by Thermogenic Adipose Tissues of Mice.
    Markus Heine, Carlotta Corban, Joerg Heeren.
      The uptake of glucose, non-esterified fatty acids, and triglycerides into brown adipose tissue is an important determinant of systemic energy metabolism, which can be studied by metabolic turnover studies using radioactive tracers in vivo. Here, we address the uptake of glucose and lipid tracers into metabolically active organs with a focus on thermogenically activated adipose tissues. Uptake by beige and brown adipocytes is highly dependent on conditions such as ambient temperature, but also varies between fasted compared to postprandial states. Accordingly, we provide methodological insights how to quantify glucose and lipid disposal under multiple physiological and environmental conditions.
    Keywords:  Chylomicrons; Glucose; Metabolic turnover studies; Triglycerides; VLDL
    DOI:  https://doi.org/10.1007/978-1-0716-2087-8_7
  4. Methods Mol Biol. 2022 ;2448 131-139
    Activation of UCP1-Independent Ca2+ Cycling Thermogenesis by Wireless Optogenetics.
    Kenji Ikeda, Kazuki Tajima, Yuji Tanabe, Ada S Y Poon, Shingo Kajimura.
      The identification of non-canonical UCP1-independent thermogenic mechanisms offers new opportunities to target such pathways to improve metabolic health. Based on our recent studies on Ca2+ futile cycling thermogenesis in beige fat, we applied the newly developed implantable wireless optogenetic system to activate Ca2+ cycling in an adipocyte-specific manner without external stimuli, i.e., fat-specific cold mimetics. Here, we describe the detailed methodology and application to the prevention of obesity.
    Keywords:  Beige fat; Ca2+ cycling thermogenesis; Obesity; Optogenetics; SERCA2
    DOI:  https://doi.org/10.1007/978-1-0716-2087-8_9
  5. Endocrinology. 2022 Feb 16. pii: bqac018. [Epub ahead of print]
    Adipose mitochondrial complex I deficiency modulates inflammation and glucose homeostasis in a sex-dependent manner.
    Kyung-Mi Choi, Karen K Ryan, John C Yoon.
      Mitochondrial dysfunction in adipose tissue has been associated with type 2 diabetes, but it is unclear whether it is a cause or the consequence. Mitochondrial complex I is a major site of reactive oxygen species generation and a therapeutic target. Here we report that genetic deletion of the complex I subunit Ndufs4 specifically in adipose tissue results in an increased propensity to develop diet-induced weight gain, glucose intolerance, and elevated levels of fat inflammatory genes. This outcome is apparent in young males but not in young females, suggesting that females are relatively protected from the adverse consequences of adipose mitochondrial dysfunction for metabolic health. Mutant mice of both sexes exhibit defects in brown adipose tissue thermogenesis. Fibroblast growth factor 21 (FGF21) signaling in adipose tissue is selectively blunted in male mutant mice relative to wild-type littermates, consistent with sex-dependent regulation of its autocrine/paracrine action in adipocytes. Together, these findings support that adipocyte-specific mitochondrial dysfunction is sufficient to induce tissue inflammation and can cause systemic glucose abnormalities in male mice.
    Keywords:  FGF21; Ndufs4; impaired glucose tolerance; inflammation; mitochondria
    DOI:  https://doi.org/10.1210/endocr/bqac018
  6. Nat Metab. 2022 Feb 14.
    Impaired phosphocreatine metabolism in white adipocytes promotes inflammation.
    Salwan Maqdasy, Simon Lecoutre, Gianluca Renzi, Scott Frendo-Cumbo, David Rizo-Roca, Thomas Moritz, Marta Juvany, Ondrej Hodek, Hui Gao, Morgane Couchet, Michael Witting, Alastair Kerr, Martin O Bergo, Robin P Choudhury, Myriam Aouadi, Juleen R Zierath, Anna Krook, Niklas Mejhert, Mikael Rydén.
      The mechanisms promoting disturbed white adipocyte function in obesity remain largely unclear. Herein, we integrate white adipose tissue (WAT) metabolomic and transcriptomic data from clinical cohorts and find that the WAT phosphocreatine/creatine ratio is increased and creatine kinase-B expression and activity is decreased in the obese state. In human in vitro and murine in vivo models, we demonstrate that decreased phosphocreatine metabolism in white adipocytes alters adenosine monophosphate-activated protein kinase activity via effects on adenosine triphosphate/adenosine diphosphate levels, independently of WAT beigeing. This disturbance promotes a pro-inflammatory profile characterized, in part, by increased chemokine (C-C motif) ligand 2 (CCL2) production. These data suggest that the phosphocreatine/creatine system links cellular energy shuttling with pro-inflammatory responses in human and murine white adipocytes. Our findings provide unexpected perspectives on the mechanisms driving WAT inflammation in obesity and may present avenues to target adipocyte dysfunction.
    DOI:  https://doi.org/10.1038/s42255-022-00525-9
  7. Methods Mol Biol. 2022 ;2448 291-306
    Analysis of Single-Cell/Nucleus Transcriptome Data in Adipose Tissue.
    Wenfei Sun.
      Adipose tissue is highly heterogeneous and plastic. Recent advances in single-cell/nucleus RNA sequencing technology have helped to study the cellular composition and dynamics of adipose tissue. In this protocol, I outline a typical workflow of analyzing single-cell/nucleus transcriptome data. Specifically, I show an example of how cellular populations are estimated and characterized from a single-nucleus RNAseq data set of frozen archived human adipose tissue.
    Keywords:  Adipose tissue heterogeneity; Data analysis; Single-cell RNA sequencing
    DOI:  https://doi.org/10.1007/978-1-0716-2087-8_19
  8. Methods Mol Biol. 2022 ;2448 217-234
    Isolation and Characterization of Human Brown Adipocytes.
    Camilla Scheele, Tora Ida Henriksen, Søren Nielsen.
      Brown adipose tissue (BAT) is a thermoregulatory fat with energy-consuming properties. The location and heterogeneity of this tissue makes it complicated to sample before and after interventions in humans, and an in vitro model for mechanistic and molecular studies is therefore of great value. We here describe a protocol for isolation of progenitors from the stromal vascular fraction of BAT biopsies obtained surgically from adult humans. We further present how these cells are differentiated in vitro and finally how they are characterized for thermogenic capacity. Methods for characterization described here include norepinephrine-induced thermogenic gene expression using qPCR; norepinephrine-induced mitochondrial uncoupling using the Seahorse XFe96 Analyzer, and norepinephrine-induced expression of UCP1 using the RNAscope® Technology.
    Keywords:  Adipogenesis; BAT in vitro model; Brown fat differentiation; Human BAT; Human brown adipocytes; Oxygen consumption rate in human brown adipocytes; UCP1 RNAscope
    DOI:  https://doi.org/10.1007/978-1-0716-2087-8_14
  9. Int J Mol Sci. 2022 Feb 08. pii: 1901. [Epub ahead of print]23(3):
    Inhibition of the Monocarboxylate Transporter 1 (MCT1) Promotes 3T3-L1 Adipocyte Proliferation and Enhances Insulin Sensitivity.
    Tracey Bailey, Ainhoa Nieto, Patricia McDonald.
      Enlarged, hypertrophic adipocytes are less responsive to insulin and are a hallmark feature of obesity, contributing to many of the negative metabolic consequences of excess adipose tissue. Although the mechanisms remain unclear, the adipocyte size appears to be inversely correlated with insulin sensitivity and glucose tolerance, wherein smaller adipocytes are insulin-sensitive and larger adipocytes develop insulin resistance and exhibit an impaired glucose uptake. Thus, pharmacological strategies aimed at regulating adipocyte hypertrophy (increase in adipocyte size) in favor of promoting hyperplasia (increase in adipocyte number) have the potential to improve adipocyte insulin sensitivity and provide therapeutic benefits in the context of metabolic disorders. As white adipose tissue can metabolize large amounts of glucose to lactate, using transcriptomics and in vitro characterization we explore the functional consequences of inhibiting monocarboxylate transporter 1 (MCT1) activity in fully differentiated adipocytes. Our studies show that the pharmacological inhibition of MCT1, a key regulator of the cellular metabolism and proliferation, promotes the re-entry of mature adipocytes into the cell cycle. Furthermore, we demonstrate that inhibitor-treated adipocytes exhibit an enhanced insulin-stimulated glucose uptake as compared with untreated adipocytes, and that this outcome is dependent on the cyclin-dependent kinase 1 (CDK1) activity. In summary, we identify a mechanism though which MCT1 inhibition improves the insulin sensitivity of mature adipocytes by inducing cell cycle re-entry. These results provide the foundation for future studies investigating the role MCT1 plays in adipocyte hyperplasia, and its therapeutic potential as a drug target for obesity and metabolic disease.
    Keywords:  MCT1; adipocytes; cell cycle; hyperplasia; insulin sensitivity; proliferation
    DOI:  https://doi.org/10.3390/ijms23031901
  10. Nat Immunol. 2022 Feb 14.
    γδ T cells monitor tissue health.
    Ipsita Subudhi, Shruti Naik.
      
    DOI:  https://doi.org/10.1038/s41590-022-01139-9
  11. Front Immunol. 2022 ;13 780839
    Modulation of Macrophage Immunometabolism: A New Approach to Fight Infections.
    Thierry Gauthier, Wanjun Chen.
      Macrophages are essential innate immune cells that contribute to host defense during infection. An important feature of macrophages is their ability to respond to extracellular cues and to adopt different phenotypes and functions in response to these stimuli. The evidence accumulated in the last decade has highlighted the crucial role of metabolic reprogramming during macrophage activation in infectious context. Thus, understanding and manipulation of macrophage immunometabolism during infection could be of interest to develop therapeutic strategies. In this review, we focus on 5 major metabolic pathways including glycolysis, pentose phosphate pathway, fatty acid oxidation and synthesis, tricarboxylic acid cycle and amino acid metabolism and discuss how they sustain and regulate macrophage immune function in response to parasitic, bacterial and viral infections as well as trained immunity. At the end, we assess whether some drugs including those used in clinic and in development can target macrophage immunometabolism for potential therapy during infection with an emphasis on SARS-CoV2 infection.
    Keywords:  SARS – CoV – 2; immunometabolism; infections; macrophage; therapeutics
    DOI:  https://doi.org/10.3389/fimmu.2022.780839
  12. Methods Mol Biol. 2022 ;2448 19-42
    Brown Fat Anatomy in Humans and Rodents.
    Georgia Colleluori, Jessica Perugini, Angelica Di Vincenzo, Martina Senzacqua, Antonio Giordano, Saverio Cinti.
      The Brown Adipose Tissue (BAT) is composed by mitochondrial rich, multilocular adipocytes, in strict topographical and functional relation with vasculature and noradrenergic nerves. Brown adipocytes are able to dissipate energy to produce heat, in a process known as non-shivering thermogenesis. Due to its contribution to energy expenditure, BAT is intensely studied for its potential to counteract metabolic diseases such as obesity, type 2 diabetes, dyslipidemia and cardiovascular diseases. BAT displays specific morphological characteristics that allow to assess its functional state. In this chapter we describe methodologies to properly dissect BAT depots, evaluate their gross anatomy, and assess its activation by light microscopy using peroxidase immunostaining and by laser scanning confocal microscopy using immunofluorescence. We also describe methodologies to study BAT ultrastructure by transmission and scanning electron microscopy, to visualize peroxidase immunostaining reactions at an ultrastructural level and to perform immunofluorescence reactions on paraffin-embedded samples, more often available in the clinical setting (due to the possibility to store them long-term) as opposed to fresh samples. The described techniques can be employed to study BAT morphology and activation in response to various stimuli (e.g., cold exposure; specific dietary composition) and in different pathological conditions (e.g., obesity; type 2 diabetes).
    Keywords:  Brown adipocyte; Brown adipose tissue; Browning; Confocal microscopy; Immunofluorescence; Immunohistochemistry; Light microscopy; Morphology techniques; Scanning electron microscopy; Transmission electron microscopy
    DOI:  https://doi.org/10.1007/978-1-0716-2087-8_2
  13. Methods Mol Biol. 2022 ;2448 83-96
    Activating Human Adipose Tissue with the β3-Adrenergic Agonist Mirabegron.
    Kong Y Chen, Robert J Brychta, Nikita S Israni, Alex Jiang, Hannah J Lea, Taylor N Lentz, Anne E Pierce, Aaron M Cypess.
      An appealing strategy for treatment of metabolic disease in humans is activation of brown adipose tissue (BAT), a thermogenic organ best visualized through 18F-FDG PET/CT. BAT has been activated to varying degrees by mild cold exposure. However, this approach can cause undesirable stress, and there remains no consensus protocol. Here, we describe standardized methods for both acute and chronic activation of BAT using the orally administered β3-adrenergic receptor (AR) agonist, mirabegron. Acute pharmacological stimulation has enabled quantification of whole-body BAT volume and metabolic activity using PET/CT imaging, and chronic stimulation increases these properties of BAT over time.
    Keywords:  18F-FDG PET/CT; Brown adipose tissue; Mirabegron; β3-adrenergic receptor agonist
    DOI:  https://doi.org/10.1007/978-1-0716-2087-8_5
  14. Mol Nutr Food Res. 2022 Feb 19. e2100944
    Zinc Levels Affect the Metabolic Switch of T Cells by Modulating Glucose Uptake and Insulin Receptor Signaling.
    Yiming Peng-Winkler, Inga Wessels, Lothar Rink, Henrike Josephine Fischer.
       SCOPE: T cell activation requires a metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis to rapidly provide substrates for biosynthesis. An individual's zinc status plays an important role in balancing the activation of T cells and is required for a proper function of immune cells. Furthermore, zinc plays an important role during effector T cell polarization to T helper cell subsets or regulatory T cells, with effector T cells relying on glycolysis and regulatory T cells on oxidative phosphorylation. Therefore, we aimed to analyze if zinc also impacts on T cell activation on the level of intracellular metabolism.
    METHODS AND RESULTS: We used mixed lymphocyte culture and anti-CD3/CD28 stimulation as in vitro models for T cell activation to investigate the effect of zinc supplementation and deprivation on metabolic switching. We observed promoted glucose uptake, insulin receptor expression and signaling in both zinc conditions, whereas key metabolic enzymes were stimulated mainly by zinc deprivation. Alterations in cytokine production suggest an immune-activating effect of zinc deprivation and a balancing effect of zinc supplementation.
    CONCLUSION: Our results suggest a supportive effect of both zinc supplementation and deprivation on the metabolic switch during T cell activation, adding another level of immune regulation by zinc. This article is protected by copyright. All rights reserved.
    Keywords:  T cell; zinc ; glycolytic switch; immunology; metabolism
    DOI:  https://doi.org/10.1002/mnfr.202100944
  15. Int J Mol Sci. 2022 Feb 01. pii: 1706. [Epub ahead of print]23(3):
    Sleep Disturbance and Metabolic Dysfunction: The Roles of Adipokines.
    Zhikui Wei, You Chen, Raghu P Upender.
      Adipokines are a growing group of peptide or protein hormones that play important roles in whole body metabolism and metabolic diseases. Sleep is an integral component of energy metabolism, and sleep disturbance has been implicated in a wide range of metabolic disorders. Accumulating evidence suggests that adipokines may play a role in mediating the close association between sleep disorders and systemic metabolic derangements. In this review, we briefly summarize a group of selected adipokines and their identified function in metabolism. Moreover, we provide a balanced overview of these adipokines and their roles in sleep physiology and sleep disorders from recent human and animal studies. These studies collectively demonstrate that the functions of adipokine in sleep physiology and disorders could be largely twofold: (1) adipokines have multifaceted roles in sleep physiology and sleep disorders, and (2) sleep disturbance can in turn affect adipokine functions that likely contribute to systemic metabolic derangements.
    Keywords:  adipokine; cardiometabolic disease; metabolic syndrome; obstructive sleep apnea; sleep disorder
    DOI:  https://doi.org/10.3390/ijms23031706
  16. Cytokine. 2022 Feb 10. pii: S1043-4666(22)00030-8. [Epub ahead of print]152 155821
    IL-21 plays an important role in modulating "Th17-Treg" cell axis in leprosy Type 1 reactions.
    Chaman Saini, Leena Sapra, Asha Bhardwaj, Mohd Tarique, Alpana Sharma, Neena Khanna, V Ramesh, Poonam Puri, Rupesh K Srivastava.
      Leprosy type 1 reaction (T1R) is a cell-mediated inflammatory reaction which involves skin and peripheral nerves in leprosy. Lesions with T1R have higher production of IL-17 cytokine from CD4+ T cells along with lower TGF-β producing FOXP3+ CD4+ Tregs. IL-21 is an important cytokine that promotes the development and stability of Th17 cells in an autocrine manner. It can play an important role in the pathogenesis of T1R in leprosy. However, the mechanism by which IL-21 influences the pathogenic progress of leprosy T1R remains poorly understood. In the present study, we evaluated the expression of IL-21 cytokine in skin lesions of both non-reactional (NR) and T1R via immuno-histochemistry and quantitative PCR (qPCR). Further, expression of various genes (IL-17A, IL-17F, TGF-β, FOXP3, RORC and IL-21) was also measured by qPCR in cultured cells. We also analyzed the secretion of various cytokines such as of IL-21, IL-17A/F and TGF-β in the culture supernatants by ELISA. In addition, differentiation of Th17 and Treg cells were studied in PBMC cultures after stimulation with Mycobacterium leprae sonicated antigens and rIL-21 for 48 hrs and the phenotypes of Th17 and Tregs were determined by flowcytometric analysis. Our results clearly indicate that IL-21+T cells were significantly higher in both peripheral blood and skin lesions of T1R as compared to NR patients. Moreover, we observed that recombinant IL-21 cytokine inhibited TGF-β producing Treg cells differentiation along with up-regulating Th17 cells under in-vitro conditions. The gene expression of IL-21 was significantly negatively correlated with Treg and positively correlated with Th17 cell markers in T1R patients. Our results suggested that IL-21 promotes T1R mediated inflammation via modulating the balance of Th17 and Treg cell populations.
    Keywords:  IL-21; Leprosy; T1R; Th17; Treg
    DOI:  https://doi.org/10.1016/j.cyto.2022.155821
  17. Methods Mol Biol. 2022 ;2448 119-130
    Stable Isotope Tracing and Metabolomics to Study In Vivo Brown Adipose Tissue Metabolic Fluxes.
    Su Myung Jung, Johnny Le, Will G Doxsey, John A Haley, Grace Park, David A Guertin, Cholsoon Jang.
      Brown adipose tissue (BAT) demonstrates extraordinary metabolic capacity. Previous research using conventional radio tracers reveals that BAT can act as a sink for a diverse menu of nutrients; still, the question of how BAT utilizes these nutrients remains unclear. Recent advances in mass spectrometry (MS) coupled to stable isotope tracing methods have greatly improved our understanding of metabolism in biology. Here, we have developed a BAT-tailored metabolomics and stable isotope tracing protocol using, as an example, the universally labeled 13C-glucose, a key nutrient heavily utilized by BAT. This method enables metabolic roadmaps to be drawn and pathway fluxes to be inferred for each nutrient tracer within BAT and its application could uncover new metabolic pathways not previously appreciated for BAT physiology.
    Keywords:  Brown adipose tissue; Brown fat; Gavage; Glucose; Liquid chromatography-mass spectrometry (LC-MS); Metabolism; Metabolomics; Stable isotope tracing; Temperature acclimation
    DOI:  https://doi.org/10.1007/978-1-0716-2087-8_8
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