bims-ciryme Biomed News
on Circadian rhythms and metabolism
Issue of 2020‒12‒13
thirteen papers selected by
Gabriela Da Silva Xavier
University of Birmingham

  1. Curr Opin Lipidol. 2020 Dec 03.
      PURPOSE OF REVIEW: The aim of this review is to present the latest findings on the role of the circadian clock in the control of metabolism, and the therapeutic potential of chronotherapy to regulate energy homeostasis in humans.RECENT FINDINGS: We summarized the recent advances related to circadian clock regulation of food intake and energy expenditure. In peripheral organs, mitochondrial oxidative capacity and lipolysis show circadian pattern in humans, and rhythms disruption may be involved in the pathogenesis of metabolic diseases. Indeed, circadian desynchrony affects food intake, insulin sensitivity, and increases the risk of developing metabolic disease. Time-targeted strategies, which aim to synchronize external cues with the molecular clock to improve metabolic outcomes, have positive effects on metabolism in humans, with several studies showing that time-targeted feeding improves body weight loss and glucose tolerance.
    SUMMARY: The interest in time-targeted strategies to prevent or manage metabolic disturbances has grown this past year with encouraging health benefits. To maximize the therapeutic effect of these strategies, further research is warranted to delineate the molecular regulation of metabolic processes controlled by the clock and especially its modulation in contexts such as aging, sex differences, or metabolic diseases.
  2. Methods Mol Biol. 2021 ;2130 233-247
      Circadian rhythms are 24-h cycles in physiology and behavior that occur in virtually all organisms. These processes are not simply driven by changes in the external environment as they persist under constant conditions, providing evidence for an internal biological clock. In mammals, this clock is located in the hypothalamic suprachiasmatic nuclei (SCN) and is based upon an intracellular mechanism composed of a transcriptional-translational feedback loop composed of a number of core clock genes. However, a clock is of no use unless it can be set to the correct time. The primary time cue for the molecular clock in the SCN is light detected by the eye. The photoreceptors involved in this process include the rods and cones that mediate vision, as well as the recently identified melanopsin-expressing photosensitive retinal ganglion cells (pRGCs). Light information is conveyed to the SCN via the retinohypothalamic tract, resulting in an intracellular signaling cascade which converges on cAMP-response elements in the promoters of several key clock genes. Over the last two decades a number of studies have investigated the transcriptional response of the SCN to light stimuli with the aim of further understanding these molecular signaling pathways. Here we provide an overview of these studies and provide protocols for studying the molecular responses to light in the SCN clock.
    Keywords:  Clock; Laser capture microdissection (LCM); Photosensitive retinal ganglion cells (pRGCs); RNA extraction; Suprachiasmatic nuclei (SCN); Transcriptional translational feedback loop (TTFL)
  3. Methods Mol Biol. 2021 ;2130 53-67
      Human cells, especially primary fibroblasts from skin punch biopsy, have emerged over the last decade as powerful, unlimited, and easily accessible resources that bridge the gap between animal models and human subjects in basic as well as clinical research. The cells also retain molecular circadian clocks that reflect subject-specific differences in circadian physiology, and the cellular rhythms can be measured easily in large scale. This is a series of protocols that describes the procedure to measure circadian rhythms in these cells, starting from deriving fibroblasts from skin punch biopsy, to generation of stable cells expressing a circadian reporter, and finally measurement of cellular rhythms in large scale.
    Keywords:  Circadian rhythm measurement; Human primary skin fibroblasts; Human skin punch biopsy; Stable cell line generation
  4. Methods Mol Biol. 2021 ;2130 195-203
      Daily rhythms of behaviors and physiologies are driven by transcriptional-translational negative feedback loops of clock genes and encoded clock proteins (Bass and Takahashi Science 330:1349-1354, 2010; Brown et al. Dev Cell 22:477-487, 2012). Posttranslational modifications of clock proteins, including protein phosphorylation, play an essential role for normal oscillation of the circadian clock through regulation of their activities, stabilities, interactions, and intracellular localization (Gallego and Virshup Nat Rev Mol Cell Biol 8:139-148, 2007; Hirano et al. Nat Struct Mol Biol 23:1053-1060, 2016). In this chapter, we describe detailed methods for quantitative analysis of phosphorylation levels of clock proteins, particularly focusing on circadian phosphorylation of CLOCK, BMAL1, and their complex (Yoshitane et al. Mol Cell Biol 29:3675-3686, 2009).
    Keywords:  Circadian rhythms; Clock proteins; Immunoblotting; Immunoprecipitation; Protein phosphorylation
  5. Methods Mol Biol. 2021 ;2130 79-85
      With the emergence of big data science, the question how we can easily collect meaningful information about circadian clock phenotypes in large human cohorts imposes itself. Here, we describe potentials and limitations of using questionnaires, specifically the Munich ChronoType Questionnaire (MCTQ), to characterize such circadian phenotypes. We also discuss scenarios when alternative methods might be more appropriate.
    Keywords:  Chronotype; Circadian disruption; Diurnal preference; Entrainment; Misalignment; Phenotyping; Proxy; Sleep; Sleep timing
  6. Methods Mol Biol. 2021 ;2130 207-219
      Live imaging of the molecular clockwork within the circadian pacemaker neurons offers the unique possibility to study complex interactions between the molecular clock and neuronal communication within individual neurons and throughout the entire circadian circuitry. Here we describe how to establish brain explants and dissociated neuron culture from Drosophila larvae, guidelines for time-lapse fluorescence microscopy, and the method of image analysis. This approach enables the long-term monitoring of fluorescence signals of circadian reporters at single-cell resolution and can be also applicable to analyze real-time expression of other fluorescent probes in Drosophila neurons.
    Keywords:  Drosophila dissociated neuronal culture; Drosophila whole brain explant culture; Time-lapse fluorescence microscopy
  7. Methods Mol Biol. 2021 ;2130 127-148
      Circadian gene transcription transmits timing information and drives cyclic physiological processes across various tissues. Recent studies indicate that oscillating enhancer activity is a major driving force of rhythmic gene transcription. Functional circadian enhancers can be identified in an unbiased manner by correlation with the rhythms of nearby gene transcription.Global run-on sequencing (GRO-seq) measures nascent transcription of both pre-mRNAs and enhancer RNAs (eRNAs) at a genome-wide level, making it a unique tool for unraveling complex gene regulation mechanisms in vivo. Here, we describe a comprehensive protocol, ranging from wet lab to in silico analysis, for detecting and quantifying circadian transcription of genes and eRNAs. Moreover, using gene-eRNA correlation, we detail the steps necessary to identify functional enhancers and transcription factors (TFs) that control circadian gene expression in vivo. While we use mouse liver as an example, this protocol is applicable for multiple tissues.
    Keywords:  Circadian; Enhancer RNA; Global run-on sequencing; Nascent RNA; Transcription factor
  8. Methods Mol Biol. 2021 ;2130 169-183
      Lipidomics has been defined as the large-scale analysis of lipids in organelles, cells, tissues, or whole organisms. Including the temporal aspects of lipid metabolic changes into this analysis allows to access yet another important aspect of lipid regulation. The resulting methodology, circadian lipidomics, has thus emerged as a novel tool to address the enormous complexity, which is present among cellular lipids. Here, we describe how mass spectrometry-based circadian lipidomics can be applied to study the impact of peripheral clocks on lipid metabolism in human primary cells and tissues, exemplified by studies in human pancreatic islets and skeletal myotubes.
    Keywords:  Circadian clock; Human pancreatic islets; Human primary myotubes; Human skeletal muscle; Lipid metabolism; Lipidomics
  9. Methods Mol Biol. 2021 ;2130 295-302
      Advances in imaging technology, combined with the development of bioluminescent reporters for core clock genes, has enabled the observation of spatiotemporal patterns of circadian rhythms in the suprachiasmatic nuclei (SCN). In particular, the PERIOD2::luciferase (PER2::LUC) knockin mouse has led to novel approaches for studying the heterogeneous circadian network in the SCN. This chapter describes how to automate the processing of PER2::LUC imaging data from SCN slices for generating spatiotemporal maps of circadian parameters like phase, period, and amplitude. These maps can be aligned and averaged to produce composite maps displaying common features across multiple slices. In addition, we describe a method for automated detection of cell-like regions of interest, to support the study of the neural network in the SCN.
    Keywords:  Automated ROI identification; Bioluminescence; Circadian oscillations; Image processing; SCN; Spatiotemporal maps
  10. Mol Cell Endocrinol. 2020 Dec 04. pii: S0303-7207(20)30410-X. [Epub ahead of print] 111108
      REV-ERBα is a nuclear receptor that inhibits Bmal1 transcription as part of the circadian clock molecular mechanism. Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a master regulator of cell and whole-body energy homeostasis, that serves as an important link between metabolism and circadian clock, in part, by regulating BMAL1 activity. While the connection of REV-ERBα to the circadian clock molecular mechanism is well characterized, the interaction between mTORC1, REV-ERBα and the circadian clock machinery is not very clear. We used leucine and rapamycin to modulate mTORC1 activation and evaluate this effect on circadian rhythms. In the liver, mTORC1 was inhibited by leucine. REV-ERBα overexpression activated the mTORC1 signaling pathway via transcription inhibition of mTORC1 inhibitor, Tsc1, antagonizing the effect of leucine, while its silencing downregulated mTORC1 signaling. Activation of mTORC1 led to increased levels of BMAL1 phosphorylation. Activation as well as inhibition of mTORC1 led to altered circadian rhythms in mouse muscle. Inhibition of liver mTORC1 by leucine or rapamycin led to low-amplitude circadian rhythms. In summary, our study shows that leucine inhibits liver mTORC1 pathway leading to dampened circadian rhythms. REV-ERBα activates the mTORC1 pathway, leading to phosphorylation of the clock protein BMAL1.
    Keywords:  REV-ERB; circadian; clock; leucine; liver; mTOR
  11. Nat Commun. 2020 12 10. 11(1): 6326
      It is well recognized that ventromedial hypothalamus (VMH) serves as a satiety center in the brain. However, the feeding circuit for the VMH regulation of food intake remains to be defined. Here, we combine fiber photometry, chemo/optogenetics, virus-assisted retrograde tracing, ChR2-assisted circuit mapping and behavioral assays to show that selective activation of VMH neurons expressing steroidogenic factor 1 (SF1) rapidly inhibits food intake, VMH SF1 neurons project dense fibers to the paraventricular thalamus (PVT), selective chemo/optogenetic stimulation of the PVT-projecting SF1 neurons or their projections to the PVT inhibits food intake, and chemical genetic inactivation of PVT neurons diminishes SF1 neural inhibition of feeding. We also find that activation of SF1 neurons or their projections to the PVT elicits a flavor aversive effect, and selective optogenetic stimulation of ChR2-expressing SF1 projections to the PVT elicits direct excitatory postsynaptic currents. Together, our data reveal a neural circuit from VMH to PVT that inhibits food intake.
  12. Methods Mol Biol. 2021 ;2130 185-193
      Recent advances in mass spectrometry (MS)-based quantitative proteomics now allow the identification and quantification of deep proteomes and post-translational modifications (PTMs) in relatively short times. Therefore, in the last few years, this technology has proven successful in the circadian field to characterize temporal oscillations of the proteome and more recently PTMs in cellular systems and in tissues. In this chapter, we describe a robust and simple protocol, based on the EasyPhos workflow, to enable preparation of large number of proteomes and phosphoproteomes from mouse tissues for MS-based quantitative analysis. We additionally discuss computational methods to analyze proteome and phosphoproteome time series to determine circadian oscillations.
    Keywords:  Circadian; Mass spectrometry; Phosphoproteomics; Proteomics
  13. Methods Mol Biol. 2021 ;2130 303-324
      In mammals, the part of the nervous system responsible for most circadian behavior can be localized to a bilaterally paired structure in the hypothalamus known as the suprachiasmatic nucleus (SCN). Understanding the mammalian circadian system will require a detailed multilevel analysis of neural SCN circuits ex vivo and in vivo. Many of the techniques and approaches that are used for the analysis of the circuitry driving circadian oscillations in the SCN are similar to those employed in other brain regions. There is, however, one fundamental difference that needs to be taken into consideration, that is, the physiological, cell, and molecular properties of SCN neurons vary with the time of day. In this chapter, we will consider the preparations and electrophysiological techniques that we have used to analyze the SCN circuit focusing on the acute brain slice and intact, freely moving animal.
    Keywords:  Biological clock; Brain slice; Circadian; In vivo electrophysiology; Neural activity rhythms; Suprachiasmatic nucleus