Rewiring of liver diurnal transcriptome rhythms by triiodothyronine (T3) supplementation

  1. Leonardo Vinicius Monteiro de Assis
  2. Lisbeth Harder
  3. José Thalles Lacerda
  4. Rex Parsons
  5. Meike Kaehler
  6. Ingolf Cascorbi
  7. Inga Nagel
  8. Oliver Rawashdeh
  9. Jens Mittag
  10. Henrik Oster

  • Curated by eLife

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    Evaluation Summary:

    Monteiro de Assis et al. demonstrate a role for T3 in modulating circadian metabolic rhythms both systemically and within the liver. The findings extend the molecular framework in which organismal metabolism is coordinated in a circadian fashion.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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Abstract

Diurnal (i.e., 24 hr) physiological rhythms depend on transcriptional programs controlled by a set of circadian clock genes/proteins. Systemic factors like humoral and neuronal signals, oscillations in body temperature, and food intake align physiological circadian rhythms with external time. Thyroid hormones (THs) are major regulators of circadian clock target processes such as energy metabolism, but little is known about how fluctuations in TH levels affect the circadian coordination of tissue physiology. In this study, a high triiodothyronine (T 3 ) state was induced in mice by supplementing T 3 in the drinking water, which affected body temperature, and oxygen consumption in a time-of-day-dependent manner. A 24-hr transcriptome profiling of liver tissue identified 37 robustly and time independently T 3 -associated transcripts as potential TH state markers in the liver. Such genes participated in xenobiotic transport, lipid and xenobiotic metabolism. We also identified 10–15% of the liver transcriptome as rhythmic in control and T 3 groups, but only 4% of the liver transcriptome (1033 genes) were rhythmic across both conditions – amongst these, several core clock genes. In-depth rhythm analyses showed that most changes in transcript rhythms were related to mesor (50%), followed by amplitude (10%), and phase (10%). Gene set enrichment analysis revealed TH state-dependent reorganization of metabolic processes such as lipid and glucose metabolism. At high T 3 levels, we observed weakening or loss of rhythmicity for transcripts associated with glucose and fatty acid metabolism, suggesting increased hepatic energy turnover. In summary, we provide evidence that tonic changes in T 3 levels restructure the diurnal liver metabolic transcriptome independent of local molecular circadian clocks.

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  1. Version published to 10.7554/elife.79405 on eLife
  2. eLife

    Author Response:

    Reviewer #3 (Public Review):

    They tried to examine the role of thyroid hormone on circadian coordination of physiological rhythmicity and hepatic gene expression, particularly under hyperthyroidism. They found that thyroid hormone status does not significantly alter the circadian rhythmicity in behavior, metabolic parameters, and gene expression pattern in the liver. For gene expression analysis, they used microarray analysis, which allowed them to analyze a large number of gene expressions simultaneously. They carefully analyzed a large number of data and the conclusion was made based on their findings.

    We thank the reviewer for the criticism and time dedicated in evaluating our manuscript.

    However, for RNA quantification, they only used data obtained by microarray analysis. From our experience, microarray data may not always be consistent with RT-PCR data. Thus, I suggest applying the RT-PCR study to several representative genes shown in Figs. 2, 3, and 4 for accuracy.

    We thank the reviewer for this comment. From our own experience microarray data usually closely replicate data from single-gene qPCR studies (see, e.g., Oster et al. Cell Metab 2006, Husse et al. 2017) and validation of every identified target by qPCR would be a very laborious task without much addition to the paper itself. Moreover, in our manuscript we were careful to not make any strong statements based on single-gene changes, but rather rely on pathway/gene group data which are expected to be more robust. Nevertheless, we now provide qPCR validation for the core clock genes presented in Fig. 3. The data (Fig. S3) and analysis show that, by and large, clock gene regulation as detected by microarray is replicated by qPCR analysis. Importantly, this holds true for the conclusion that high T3 phase delays the liver clock without further effects.

    Along the same line, performing Western blot analysis of several genes may be also important to perform. Particularly, because they claimed that the time-dependent effect of T3 could be due to rhythmicity in TH transporters, Dol 1, and TH receptor expression and/or activity, protein analysis of activity analysis of such factors is strongly recommended.

    Please also see our reply to the point above (qPCR validation). We agree with the reviewer’s point on potential further levels of TH regulation. Analyzing TH transport and DIO1 activity around the clock would potentially provide additional explanations for the circadian modulation of T3 action, and it would be interesting to study potential changes in these phenomena under different T3 concentrations. However, testing these on the functional level would require an extensive amount of time and potentially lead to further follow-up experiments far beyond the scope of this manuscript. Following the guidelines of eLife, though, we have fully acknowledged this limitation in our manuscript.

    Lines 494 – 498 “One limitation of our finding is the lack of data on the diurnal regulation of T3 effects at the protein level and on enzymatic regulation. It is not unlikely that these represent additional ways by which circadian rhythms and TH action can interact. Our conclusions arise from gene expression data and, therefore, may not fully account for the full spectrum of diurnal modulation of TH action in the livers”.

  3. eLife

    Evaluation Summary:

    Monteiro de Assis et al. demonstrate a role for T3 in modulating circadian metabolic rhythms both systemically and within the liver. The findings extend the molecular framework in which organismal metabolism is coordinated in a circadian fashion.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Monteiro et al. sought to determine the role of various thyroid hormones (T3 and T4) in supporting circadian rhythmicity. The authors achieved this through gain of function experiments (T3) supplementation as well as comprehensive metabolic and transcriptional profiling. The authors find that T3 supplementation recapitulates a variety of manifestations of hyperthyroidism such as increased body temperature and increased food and water intake. Furthermore, the authors found that T3 supplementation leads to alterations in hepatic metabolic genes, ultimately culminating in alterations in glucose and lipid metabolism. The data generated in this manuscript will serve the field of circadian rhythm biology by providing an additional transcriptomic atlas of hepatic alterations during both times of day as well as in response to T3 supplementation.

  5. eLife

    Reviewer #2 (Public Review):

    The manuscript "Rewiring of liver diurnal transcriptome rhythms by triiodothyronine (T3) supplementation" by Dr. Leonardo Vinícius Monteiro de Assis, Dr. Lisbeth Harder, and colleagues addresses the impact of elevated triiodothyronine (T3) levels in the regulation of liver diurnal transcriptional rhythms. The authors induced hyperthyroidism in mice by supplementing the drinking water with T3. In-depth circadian analysis of metabolic and behavioral rhythms revealed that such T3 supplementation profoundly influences metabolism in a diurnal manner. Furthermore, the authors provide a large-scale analysis of the liver transcriptional landscape conducted around the clock in T3-treated animals and control counterparts by microarray. These analyses provide novel significant insights into the impact of thyroid hormones on the diurnal regulation of the liver transcripts and allowed dissection, to some extent, between T3-dependent and T3-independent regulation of liver circadian transcriptome.

  6. eLife

    Reviewer #3 (Public Review):

    They tried to examine the role of thyroid hormone on circadian coordination of physiological rhythmicity and hepatic gene expression, particularly under hyperthyroidism. They found that thyroid hormone status does not significantly alter the circadian rhythmicity in behavior, metabolic parameters, and gene expression pattern in the liver. For gene expression analysis, they used microarray analysis, which allowed them to analyze a large number of gene expressions simultaneously. They carefully analyzed a large number of data and the conclusion was made based on their findings.

    However, for RNA quantification, they only used data obtained by microarray analysis. From our experience, microarray data may not always be consistent with RT-PCR data. Thus, I suggest applying the RT-PCR study to several representative genes shown in Figs. 2, 3, and 4 for accuracy. Along the same line, performing Western blot analysis of several genes may be also important to perform. Particularly, because they claimed that the time-dependent effect of T3 could be due to rhythmicity in TH transporters, Dol 1, and TH receptor expression and/or activity, protein analysis of activity analysis of such factors is strongly recommended.

  7. Version published to 10.1101/2022.04.28.489909v1 on bioRxiv