Wu et al. BMC Molecular and Cell Biology(2024) 25:11
https://doi.org/10.1186/s12860-024-00508-6

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

Mice lacking DIO3 exhibit sex-specificalterations in circadian patternsof corticosterone and gene expression
in metabolic tissues
Zhaofei Wu1* , M. Elena Martinez1 and Arturo Hernandez1,2,3
Abstract
Disruption of circadian rhythms is associated with neurological, endocrine and metabolic pathologies. We haverecently shown that mice lacking functional type 3 deiodinase (DIO3), the enzyme that clears thyroid hormones,
exhibit a phase shift in locomotor activity, suggesting altered circadian rhythm. To better understand thephysiological and molecular basis of this phenotype, we used Dio3+/+ and Dio3-/- mice of both sexes at different
zeitgeber times (ZTs) and analyzed corticosterone and thyroxine (T4) levels, hypothalamic, hepatic, and adipose
tissue expression of clock genes, as well as genes involved in the thyroid hormone action or physiology of liver
and adipose tissues. Wild type mice exhibited sexually dimorphic circadian patterns of genes controlling thyroid
hormone action, including Dio3. Dio3-/- mice exhibited altered hypothalamic expression of several clock genes
at ZT12, but did not disrupt the overall circadian profile. Expression of clock genes in peripheral tissues was not
disrupted by Dio3 deficiency. However, Dio3 loss in liver and adipose tissues disrupted circadian profiles of genesthat determine tissue thyroid hormone action and physiology. We also observed circadian-specific changes inserum T4 and corticosterone as a result of DIO3 deficiency. The circadian alterations manifested sexual dimorphism.
Most notable, the time curve of serum corticosterone was flattened in Dio3-/- females. We conclude that Dio3
exhibits circadian variations, influencing the circadian rhythmicity of thyroid hormone action and physiology in liver
and adipose tissues in a sex-specific manner. Circadian disruptions in tissue physiology may then contribute to themetabolic phenotypes of DIO3-deficient mice.
Keywords Thyroid hormone action, Type 3 deiodinase, Type 2 deiodinase, Circadian rhythm, Clock genes,
Corticosterone, Hypothalamus, Liver, Adipose tissue*Correspondence:
Zhaofei Wu
Zhaofei.Wu@mainehealth.org
1
MaineHealth Institute for Research, MaineHealth,04074 Scarborough, ME,, USA
2
Department of Medicine, Tufts University School of Medicine,
02111 Boston, MA, USA
3
Graduate School of Biomedical Sciences and Engineering, University ofMaine, 04469 Orono, Maine, USA© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Wu et al. BMC Molecular and Cell Biology(2024) 25:11

Introduction
Adequate regulation of circadian rhythms is critical forneurological and metabolic health. In humans, circadiandysregulation has been associated with multiple neurological [1–5] and metabolic [6–8] pathologies, underscoring the importance of circadian patterns in physiological
functions for the maintenance of health. Some of thesefunctions include endocrine systems, of which the
hypothalamic-pituitary-adrenal axis and glucocorticoid hormones are the most representative example, as
glucocorticoid levels in rodents and humans manifest a
pronounced circadian profile, with highest levels at thetransition into the active cycle [9].
In the context of circadian biology, the physiology ofthyroid hormones has been less studied, but scatteredobservations in different tissues and models suggest theirrelevance. In humans, serum levels of thyroxine (T4),the main thyroid hormone and largely considered a prohormone, show circadian variations and abnormalities
in depressive patients [10]. In the rat, concentrations oftriiodothyronine (T3), the active thyroid hormone, havebeen shown to follow a circadian variation in the brain
and liver of the rat [11, 12]. In the chicken, the expressionof Thsb, a gene critical for the synthesis of thyrotropin(TSH), which controls thyroid gland function, exhibits
diurnal variations [13].
Critical determinants of thyroid hormone availability and action in tissues have also been shown to follow circadian patterns. For example, the hypothalamic
expression of type 2 and 3 deiodinases (DIO2 and DIO3,
respectively), the enzymes that regulate the activation or
inactivation of thyroid hormones in target tissues, areabnormal in chickens exposed to different photoperiods[13]. In the rat pineal gland, a critical tissue for the production of melatonin and the regulation of sleep/awake
cycles, DIO2 activity exhibits a robust circadian pattern[14], and this is associated with circadian variations of
thyroid hormone ratios in this tissue [15]. We have previously shown that DIO3 expression in the mouse adrenal
gland exhibits a strong circadian pattern [16]. The expression of the beta isoform of the T3 receptor (Trb) also
exhibits a strong diurnal variation in the rat liver [17].
Considering the increasing appreciation of the major
contribution of deiodinases in the control of T3 action at
the local level [18–20], these observations support a substantial role for regulation of T3 action as an integral partof circadian physiological processes.
In a recent study, we have reported that Dio3-/- miceof both sexes exhibit an abnormally prolonged dark cycle,
as assessed by their locomotor activity [21], suggestingthat DIO3 deficiency causes circadian dysregulation. To
understand the underlying effects of DIO3 on circadianrhythmicity, in the present work we investigated patternsof tissue expression of genes related to the biologicalPage 2 of 14

clock and to thyroid hormone action. We observed alterations in the circadian expression patterns of these genes
in a manner that is tissue and sex-specific. These results
indicate that T3 action is regulated in a circadian manner and that circadian disruptions may contribute to theneurological, neuroendocrine and metabolic abnormalities of Dio3-/- mice.

Materials and methods
Experimental animals

Mice carrying an inactivating mutation on the Dio3 gene
(Dio3-/- mice) have been characterized before [22] andthe genotype of experimental animals was determined aspreviously described [22]. Dio3+/+ and Dio3-/- mice usedin the experiments were four months old littermates on
a 50/50 mixed C57BL/6J /129/SvJ genetic backgrounds.
Experimental and control groups thus share exactly thesame genetic background and differed only in the Dio3
mutation. They were generated by heterozygous crossesof C57BL/6J males and 129/SvJ females, and belonged
to litters of 6–9 pups in size. Animals were kept under a12-h light cycle and fed ad libitum. Animals were euthanized by asphyxiation with CO2. Blood was taken fromthe inferior vena cava and, after coagulation of 3–4 h at
4 °C, serum was obtained by centrifugation and stored
at -70 °C until use. Fresh tissues were harvested and frozen on dry ice and stored at -70 °C until further use. All
animal procedures were approved by the MaineHealthInstitute for Research Institutional Animal Care and Use
Committee.
Hormone determinations

Total serum T4 and T3 were determined in technical duplicates using the total T4 and T3 Coat-a-Count
radioimmunoassay kits from Diagnostics Products Corp.
(Los Angeles, CA, USA) according to the manufacturer’s
instructions. Serum corticosterone was determined using
the AssayMax™ ELISA Kit from Assaypro. (St Charles,
MO, USA) according to the manufacturer’s instructions.
Metabolic studies

For metabolic and physical activity studies we used metabolic cages (Promethion metabolic monitoring cage system, Las Vegas, NV). A standard 12 h light/dark cycle
was maintained throughout the calorimetry studies.
Prior to data collection, all animals were acclimated torunning wheels for 2 days. The calorimetry system consists of 16 metabolic cages (identical to home cages withbedding) each equipped with water bottles and food hoppers connected to load cells for food and water intake
monitoring, and all animals had ad libitum access to standard rodent chow and water throughout the study. All
cages contained running wheels (4.5″ (11.5 cm) diameter,Wu et al. BMC Molecular and Cell Biology(2024) 25:11

MiniMitter, Bend, OR) wired to record revolutions/second continuously using a magnetic reed switch.
Gene expression

Total RNA was isolated from mouse tissues using the
RNeasy standard or lipid tissue Mini Kits from Qiagen(Valencia, CA). Total RNA (1 µg) was reverse transcribedwith M-MLV reverse transcriptase in the presence ofrandom decamers (both from Thermo Fisher Scientific, Waltham, MA) at 65 °C for 5 min, then 37 °C for
50 min. The 20 µl reverse transcription reactions were
DNAse treated and diluted by adding 230 µl of RNase
free water. An aliquot of each sample was mixed together
for an internal standard and diluted fourfold. Real-time
PCR reactions were set up in duplicate determinations
with gene-specific primers and SYBR Select Master Mix(Thermo Fisher Scientific, Waltham, MA), and run on
the CFX Connect from Bio-Rad (Hercules, CA), where
they underwent an initial 10 min denaturing step, followed by 36 cycles of a denaturing step (94 °C for 30 s)
and an annealing/extension step (60 °C for 1 min). For
each individual sample, expression was corrected by theexpression of housekeeping gene Actb, which did notexhibit any significant difference in expression between
experimental groups (see Results). Expression data areshown in arbitrary units and represented as fold-changeover the mean value in the corresponding control group.
The sequences of the primers used for each gene determination are shown in Supplementary Table 1. Primer
sequences used in this study.
Immunofluorescence (IF)Male mice were anesthetized and perfused with ice-cold
PBS and 4% PFA through the left heart ventricle. The
perfused adrenal glands were removed, fixed for 1 h in
4% PFA, transferred to a 30% sucrose PBS solution, andstored at 4 °C. Once the glands sank to the bottom of thevials, they were frozen, embedded in OCT, and stored at− 20 °C. The 10 μm sections were prepared using a Leica
cryostat, and Cyp11b1 IF was performed as previously
described [23]. IF images were taken using the Leica SP8
confocal microscope system.
Statistical analyses

We used five or six mice per experimental group, sex
and circadian time. For specific determinations, thisnumber may have been reduced, as indicated in the corresponding Figure legend. Given the established sexual
dimorphisms in most of the parameters examined, data
for males and females were analyzed separately. Statistical analyses were performed using the statistical toolsof GraphPad Prism 6 (GraphPad Software, Inc.). A Student’s t-test, or a one-way ANOVA followed by Tukey’s
test, respectively, was used to determine statistical

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significance between two or more groups. Statistical significance was defined as P < 0.05. Unless otherwise stated,bars or lines represent the mean ± SEM. We used RStudio Pro 2022.12.0 Build 353.pro20 with R 4.2.1 and CircaCompare for circadian parameter estimation. Image J
1.53q was used to quantify fluorescence intensity for the
IF staining.

Results
Delayed circadian phase of wheel running activity

We first determined if the mice used in the present
study recapitulated that circadian alteration previously
reported [21]. We compared circadian parameters using
the recently developed statistical software CircaCompare[24]. Analysis of the running wheel results reveals statistical significance in differences in phase between Dio3+/+and Dio3-/- groups of both sexes. Results of male havestatistical significance in all three rhythmic parameters(mesor, amplitude and phase) between the two groups.
The average circadian phase of Dio3-/- mice delayed from1.08 (4.13 h, female) to1.33 (5.08 h, male) radians (Fig. 1).
However, there are no significant differences in the circadian phase of energy expenditure results between the twogroups when the running wheels were removed, althoughother two rhythmic parameters (mesor and amplitude)
still have significance in differences (male: P-value formesor difference < 0.001, P-value for amplitude difference < 0.001, P-value for difference in phase 0.37; female:
P-value for mesor difference 0.40, P-value for amplitudedifference < 0.01, P-value for difference in phase 0.057)
between the two groups (Supplementary Fig. 1). Theseobservations were consistent with the circadian phenotype previously observed in the Dio3-/- mice and supported the molecular studies included herein.
Circadian variations in serum thyroid hormones andcorticosterone

We determined serum concentrations of thyroid hormones and corticosterone at four time points (ZTs,
Zeitgeber time) (Fig. 2). Dio3+/+ male mice exhibit amarked circadian pattern of serum corticosterone, andthis rhythm was not altered in Dio3-/- males (Fig. 2a,left). Concerning serum T4, a moderate circadian pattern was observed in Dio3+/+ males with a peak value
around ZT6-ZT12. At ZT6, Dio3-/- males manifested
significantly lower serum T4, while T4 was at same levelat other ZTs (Fig. 2a, right).
A circadian pattern in serum corticosterone was also
observed in Dio3+/+ females (Rhythmic, P = 0.044), witha peak value at ZT12-ZT18. However serum corticosterone in Dio3-/- females was significantly lower than inwild-type females at ZT 12 and ZT18 (Fig. 2b, left), andits rhythm was not significant (not rhythmic, P = 0.30, allrhythmic parameters can be accessible in supplementary

Wu et al. BMC Molecular and Cell Biology(2024) 25:11

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Fig. 1 Circadian parameters of running wheel data of adult Dio3-/- mice. Typical 48 h of data was analyzed using CircaCompare, according to Parsons etal., 2020 [23]. (a), male, n = 13,13; (b), female, n = 11,11

Fig. 2 Circadian variation in serum corticosterone and T4 in adult mice. (a), Serum corticosterone and T4 in adult males. (b), Serum corticosterone andT4 in adult females. Values are expressed relative to values at ZT0 in wild type mice. *, **, ***, P < 0.05, 0.01 and 0.001, respectively, Dio3+/+ vs. Dio3-/-,as
determined by two-way ANOVA and Tukey’s post hoc test (n = 4–5). (c), Immunofluorescence of cyp11b1 in female adrenal gland. Each picture set represents the results of three micerhythmic parameters file). Similarly, serum T4 in Dio3+/+
females changed along the ZTs with a peak value at ZT18,
while T4 rhythm seems disturbed in Dio3-/- females,
and were significantly lower than those in Dio3+/+ littermates at ZT0, ZT6 and ZT18 (Fig. 2b, right). Taken
together, these observations suggest that DIO3-deficiency has significant effects on the circadian physiology

of both the HPT and HPA axes, with a remarkable sexual
dimorphism.
Adrenal gland expression (IF) of Cyp11b1

The alterations in serum corticosterone, especially infemale Dio3-/- mice, suggested dramatic changes in its
synthesis. Cyp11b1 (Steroid 11-Beta-Hydroxylase) is key

Wu et al. BMC Molecular and Cell Biology(2024) 25:11

enzyme for adrenal gland to produce corticosterone [25,26], and we analyzed its expression in the adrenal glandby immunofluorescence (IF). IF signal of Cyp11b1 at
ZT12 showed a substantial decrease in the female adrenal gland, suggesting a critical limitation in the production of corticosterone at the peak of its cycle.
Hypothalamic gene expression

We then measured hypothalamic expression of clockrelated genes at different ZTs. Clock genes were particularly impacted by DIO3 deficiency at the start of thedark cycle (ZT12), with Dio3-/- males exhibiting significant decreases in the expression of Dbp, Per1, Per2,
Clock, Tbx3, Rora and Cry2. The expression of Per2 wasincreased at ZT0 and ZT6 in the hypothalamus of Dio3/- males. We observed no significant differences in theexpression of Bmal1. (Fig. 3a)
In Dio3-/- females, hypothalamic expression of clock
genes was less affected than in males. Dio3-/- females
showed increased expression of Dbp and Tbx3 at ZT0and ZT18, of Rora and Per2 at ZT0). They also manifested decreased expression of Rora at ZT18 and of Cry2
at ZT12. We observed no significant differences in theexpression of Clock, Bmal1 and Nr1d1. (Fig. 3b)
We also investigated the expression of genes related toT3 action, including thyroid hormone receptors alphaand beta (Tra and Trb, respectively), T3-regulated genes
Hr and Klf9, and thyroid hormone deiodinases Dio2 andDio3. (We can measure Dio3 mRNA in Dio3-/- micesince these mice carry a triple point mutation in the
Dio3 coding region that renders the enzyme fully inactive [22], but leaves the mRNA largely intact). In Dio3+/+
male mice, Dio2 expression showed a pattern with higher

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expression at ZT12 and ZT18 compared to ZT0 and
ZT6. This pattern was disrupted in Dio3-/- mice, showing a substantial reduction in Dio2 expression at ZT12
(Fig. 4a). Hypothalamic Dio3 mRNA did not vary acrossthe ZTs in Dio3+/+ males, but it was markedly elevatedin Dio3-/- at ZT6, ZT12 and ZT18 (Fig. 4a). In contrast
to males, Dio2 expression was significantly increased
at ZT0 in Dio3-/- females versus controls (Fig. 4b). In
Dio3+/+ females, Dio3 mRNA showed a moderate circadian pattern, with highest expression at ZT18. Similarly
to males, Dio3 expression in Dio3-/- females was substantially increased at all ZTs (Fig. 4b).
Concerning the hypothalamic expression of the T3
receptors, Dio3-/- females showed no differences in theexpression of Tra or Trb at any ZT, but their expression,
especially that of Trb, showed a noticeable circadian profile, with a trough at ZT18 (Fig. 4b). In Dio3+/+ males,both Tra and Trb showed a circadian profile with atrough at ZT12, while Dio3-/- males showed a significant
decreased in Tra and Trb expression at ZT0 and ZT12,
respectively(Fig. 4a).
Expression of T3-regulated genes Hr and Klf9 revealedconsistent circadian patterns same as Dio2 or Dio3 in
Dio3+/+ males or females, respectively (Fig. 4), but weobserved significant differences in Dio3-/- mice. In Dio3/- females, Hr expression was increased at ZT0 and ZT18
in Dio3-/-, while Klf9 expression was increased at ZT18
(Fig. 4b). In Dio3-/- males, there was no significant differences in the expression of Klf9, but Hr mRNA was
increased at ZT0 and decreased at ZT12 (Fig. 4a). These
results suggest T3 drove the phase shift in gene expression through T3-responsive genes.

Fig. 3 Hypothalamic expression of genes related to the circadian clock in male (A) and female (B) mice. Data represent the mean ± SEM of 4–5 mice perexperimental group and ZT. *P < 0.05, **P < 0.01, ***, P < 0.001, as determined by two-way ANOVA and Tukey’s post hoc test

Wu et al. BMC Molecular and Cell Biology(2024) 25:11

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Fig. 4 Hypothalamic expression of genes related to thyroid hormone action. (a, b) Hypothalamic gene expression in male (a) and female (b) mice. Data
represent the mean ± SEM of 4–5 mice per experimental group and ZT. *P < 0.05, **P < 0.01, ***, P < 0.001, as determined by two-way ANOVA and Tukey’s
post hoc test

We analyzed the phase shift in all the hypothalamic
gene expression results with CircaCompare. It gives a
Presence of rhythmicity (P-value) 0.0014 for Dio3-/- and0.0015 for Dio3+/+, φ1 = 0.408 (1.56 h) for the males.
However both groups of data are arrhythmic for thefemales: presence of rhythmicity (P-value) 0.26 for Dio3/- and 0.11 for Dio3+/+, φ1= -0.832 (-3.18 h). These datasuggest sex-biased alterations in the hypothalamic circadian rhythm of Dio3-/- mice.
Hepatic gene expression

We examined gene expression in the liver. In this tissue,
clock-related genes exhibited circadian patterns in wild
type mice of both sexes (Supplementary Figs. 2, 3). In
Dio3-/- males, expression of clock-related genes showedno remarkable differences (Supplementary Fig. 2). Dio3/- females showed limited differences in the hepatic
expression of clock genes, although decreased expressionof Per1 at ZT0 and Tbx3 at ZT18 seems to exacerbate thecircadian profile of these genes (Supplementary Fig. 3).
Concerning the expression of genes involved in T3
signaling, hepatic Dio3 showed a mild circadian profile
in males, with lowest expression at ZT18. This profile
was ameliorated in Dio3−/− males due to significantly

increased Dio3 expression at ZT12 and ZT18 (Fig. 5a).
T3-regulated genes Dio1, Klf9 and Hr showed rhythmic expression profiles, and the expression of T3-regulated genes Dio1 and Hr was significantly lower at ZT18
(Fig. 5a), suggesting lower T3 action at this ZT. But weobserved no circadian expression pattern in Tra or Trb.
In females, we observed a mild circadian pattern in theexpression of Dio3, Tra and Trb, with lowest expression
at ZT18 for all three genes (Fig. 5b). In Dio3-/- females,
this pattern was partially disrupted for Dio3 and Tra
due to significantly decreased expression of these genes
at ZT0. Interestingly, the hepatic expression of T3-regulated genes showed a more pronounced circadian patternin females with highest expression at ZT18 for Dio1 andHr, and at ZT6 for Klf9. However, this circadian pattern
was completely ablated for Dio1 due to significant reductions in expression at ZT12 and ZT18 in Dio3-/- females(Fig. 5b).
We also analyzed the circadian phase shift in thehepatic gene expression results. The results from malemice indicate a presence of rhythmicity (P-value) 0.068
for Dio3-/- and 0.063 for Dio3+/+, φ1 = 0.045 (0.17 h).
Both groups of data are more arrhythmic for the females:presence of rhythmicity (P-value) 0.11 for Dio3-/- andWu et al. BMC Molecular and Cell Biology(2024) 25:11

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Fig. 5 Hepatic expression of genes related to thyroid hormone action in male (a) and female (b) mice. Data represent the mean ± SEM of 4–5 mice perexperimental group and ZT. *P < 0.05, **P < 0.01, ***, P < 0.001, as determined by two-way ANOVA and Tukey’s post hoc test

0.13 for Dio3+/+, φ1 = 0.044 (0.168 h). These data indicate
no significant effect on hepatic circadian rhythm in Dio3/- of either sex.
Gene expression in white adipose tissue (WAT)The WAT expression of most clock-related genes exhibited stronger circadian profiles in females of both
genotypes (Supplementary Fig. 5) than in males (Supplementary Fig. 4). The loss of Dio3 function did not erase
the rhythm of gene expression in WAT, although wenoted a significant increase in the expression of Nr1d1
at ZT0 in the Dio3-/- male WAT. (Supplementary Fig. 4).
Similarly, although significant differences were observedin the expression of Per1 at ZT0, Bmal1 at ZT12, andNr1d1 at ZT18, these differences did not translate into amajor disruption of the overall circadian profiles of thosegenes in the WAT of females (Supplementary Fig. 5).
Concerning genes related to T3 signaling, we observedthat Dio3 did not show a circadian pattern of expression in the WAT of Dio3+/+ males, but its expression
was significantly increased in Dio3-/- male mice at all
ZTs (Fig. 6a). Interestingly, Dio2 expression, despite
the variability in the data, suggests a circadian patternof expression with highest levels at ZT18 (Fig. 6a). This
Dio2 rhythm was ablated in Dio3-/- males. In addition,
our data show that the Dio2 expression curve matchesWu et al. BMC Molecular and Cell Biology(2024) 25:11

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Fig. 6 Expression of genes related to thyroid hormone action in male (a) and female (b) mice WAT. Data represent the mean ± SEM of 4–6 mice perexperimental group and ZT. *P < 0.05, **P < 0.01, as determined by two-way ANOVA and Tukey’s post hoc test

the trend of Trb, and the Dio3 expression curve matches
the trend of Tra and Klf9 (Fig. 6a). We also measure theexpression of Lep, Lpl and Mest, which are related to adiposity [27–29]. Interestingly, the expression of Lep Lpl
and Mest showed a peak at ZT18, and this pattern was
disrupted in the WAT of Dio3−/− males (Fig. 7a). The Lep
expression was significantly decreased in Dio3-/- males
at ZT18. On the contrary, the Lpl expression was significantly increased in Dio3-/- males at ZT0 and ZT18
(Fig. 7a).
We obtained different results in the WAT of females.
In contrast to males, the expression curves all matchone similar trend in the WAT of females, which showed
peak expressions at ZT6. This pattern was disrupted in
Dio3-/- females largely as a result of a marked reduction in expression at ZT6 (Fig. 6b). We did not observe

significant alterations in Dio3-/- females in the expression of Tra, Trb, Klf9, Lep, Lpl or Mest (Figs. 6b and 7b).
However, in the case of Lep, Lpl and Mest, the trend was
similar to that of males, with peak expression at ZT18
(Fig. 7b).
We then analyzed the circadian phase shift in the WAT
gene expression results with CircaCompare. The analyses showed a presence of rhythmicity (P-value) 0.34 for
Dio3-/- and 0.13 for Dio3+/+, φ1 = 0.178 (0.68 h) for the
males. Both groups of data are more rhythmic for thefemales: presence of rhythmicity (P-value) 0.11 for Dio3/- and 0.055 for Dio3+/+, φ1 = 0.234. (0.89 h). These datasuggest Dio3-/- affects WAT circadian rhythm in both
sexes of mice.

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Fig. 7 Expression of genes related to adiposity in male (a) and female (b) mice WAT. Data represent the mean ± SEM of 4–6 mice per group and ZT.
*P < 0.05, **P < 0.01, as determined by two-way ANOVA and Tukey’s post hoc test

Gene expression in male brown adipose tissue (BAT)We extended our investigations of circadian gene expression to male BAT. In this tissue, most clock-related
genes showed a strong circadian pattern of expression.
We observed significant differences in the expression ofcertain genes (Dbp, Nr1d1) at particular ZTs (Supplementary Fig. 6), but these changes did not substantiallydisrupt the circadian pattern except for Cry2, whose
expression was significantly reduced at ZT6 and markedly increased at ZT18 (Supplementary Fig. 6).
Concerning T3-signaling, both Dio2 and Dio3 expression showed a strong circadian pattern of expression inwild type mice, with peak expression at ZT6. This pattern
was disrupted in Dio3-/- mice due to a sharp reduction
in expression precisely at ZT6 for both Dio2 and Dio3,
and to a significant expression increase at ZT18 for Dio3
(Fig. 8). Both Tra and Trb exhibited an expression pattern in males, with the lowest expression at ZT18, but theoverall pattern was not dramatically disrupted despite
small but statistically significant expression differences(Fig. 8). We observed no changes in the circadian expression of Klf9, although Hr mRNA exhibit a circadian profile with lowest expression at ZT18, a time in which theexpression was increased in Dio3-/- mice (Fig. 8).
We also analyzed the expression of genes of relevanceto BAT physiology. Pronounced circadian patterns wereobserved in Dio3+/+ mice in the expression of Pgc1a,
Prdm16 and Elovl3, with peak expression at ZT6 (Pgc1aand Prdm16) or ZT18 (Elovl3) (Fig. 9a). For Pgc1a, theexpression pattern was significantly disrupted in Dio3-/due to decreased expression at ZT6. In the case of Elovl3,
marked increases (ZT0 and ZT12) or decreases (ZT18)
in expression changed its rhythmic pattern from thecontrol (Fig. 9a). Adiponectin, Lpl, and Pparg all exhibited
significant expression pattern changes in mutant mice
(Fig. 9a). In contrast to WAT, the BAT expression of Lep
and Mest showed more significant increases in mutant
mice (Fig. 9a). The expression of Lep in Dio3-/- mice
was reduced at ZT12 and markedly elevated at ZT18.
The notable increased expression of both Lep and Mestin the BAT of mutant mice at ZT18 stands in sharp contrast with the results from WAT, in which we observed
the opposite regulation (Fig. 7a). Although we noted nosignificant change in Ucp1 expression, the expression
curve of mutant mice did show a more flattened pattern(Fig. 9a).
We also analyzed the circadian phase shift in BATgene expression data. We obtained a presence of rhythmicity (P-value) 0.04 for Dio3-/- and 0.01 for Dio3+/+,
φ1 = 0.552 (2.11 h) for the circadian clock genes (Fig. 9b).
Interestingly, there is a significant change in the rhythmicity of the lipid metabolism genes of the Dio3-/- BAT.
The analysis of the lipid metabolism genes shows the circadian rhythm was significant in the Dio3+/+ BAT anderased (Fig. 9b) in the Dio3-/- BAT: presence of rhythmicity (P-value) 0.54 for Dio3-/- and 0.02 for Dio3+/+,
φ1 = 1.211. (4.63 h). These data suggest that loss of DIO3
function in male mice eliminates the circadian rhythm oflipid metabolism in BAT, independently of clock genes,in addition to its phase shift effect on circadian genes.

Discussion
Circadian rhythms have been found in humans andalmost all organisms studied [30]. The proper coordination of these rhythms, both within the body andwith respect to the environment, is critical to health. In

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Fig. 8 Expression of genes related to thyroid hormone action in BAT of male mice. Data represent the mean ± SEM of 4–5 mice per experimental group
and ZT. *P < 0.05, **P < 0.01, ***, P < 0.001, as determined by two-way ANOVA and Tukey’s post hoc test

humans, disruptions in circadian patterns are associated with neurological conditions [10, 31–34] and maylead to metabolic disorders and increased susceptibility
to other pathologies. Scattered evidence in the literatureshows evidence of circadian patterns in the physiology
and action of thyroid hormones [12]. For example, investigators have shown a strong circadian pattern of Dio2 in
the pineal gland and pituitary [14, 35]. Given the importance of these organs in sleep cycles and neuroendocrine
regulation, this work suggests a role for thyroid hormone
action in normal tissue circadian processes. We have also
shown that DIO3 enzymatic activity exhibits a circadianpattern in the adrenal gland [16], and mice with an inactivating mutation in Dio3 manifest reduced sleep time andprolonged periods of night time physical activity [21],suggesting that Dio3 is an integral part of the cellular andtissue processes that follow a circadian rhythm. Here we
examined circadian patterns of serum hormones and tissue expression of clock- and thyroid hormone-related
genes to evaluate how DIO3 deficiency impacts those
endpoints.
In wild type mice, we observed pronounced sexual
dimorphisms in some of the parameters measured. Notably, we observe a shift between males and females in thecircadian pattern of serum corticosterone. Althoughfemales have been historically understudied [36], some
reports have described evidence of circadian shift
between males and females [37–40].

Wu et al. BMC Molecular and Cell Biology(2024) 25:11

Page 11 of 14

Fig. 9 Expression of genes relevant to BAT physiology. (a), real-time PCR data of lipid metabolism genes in BAT. Data represent the mean ± SEM of 4–5
mice per experimental group and ZT. *, **, ***, **** indicate P < 0.05, 0.01, 0.001, 0.0001, as determined by two-way ANOVA and Tukey’s post hoc test. (b),plot of the real-time PCR data using circadian analyzing software CircaCompare

Other circadian patterns of expression are highly consistent between males and females in wild type mice,
including not only that of clock-related genes but alsothat of genes pertaining to T3 signaling. For instance, inmales, the hepatic expression patterns of Dio1, a deiodinase that generates T3 from T4, and of T3 targets Klf9 and
Hr closely resemble that in females. Especially intriguingis the marked increased in the expression of T3-target
gene Hr at ZT18 but the mild peak of Klf9 expression at
ZT6 (Fig. 5). The divergence in peak time of these two
T3-regulated genes suggests their T3-regulation relies on
different mechanisms or possibly occurs in different liver
cell types. Two recent publications demonstrate that T3
supplementation induced hyperthyroid or MMI induced
hypothyroid condition can change circadian gene expression in the liver of mice [41, 42]. Consistent with theseobservations, our results also suggest T3 regulated liver
metabolism independent of local molecular circadianclocks.
Concerning Dio2 and Dio3, they exhibit circadian patterns of expression, which is consistent with previous
observations [14, 16], but those patterns are sexuallydimorphic and tissue-specific. Particularly striking in thisregard is the pronounced and identical circadian profile
of Dio2 and Dio3 expression in male BAT. The concurrence of peak expression of both enzymes suggests anenhancement of both activation and inactivation of thyroid hormone at the same time. This initially puzzlingobservation is likely explained by previous observations
that Dio2 is predominantly expressed in mature brown
adipocytes, while Dio3 expression is associated with BAT
cell progenitors [43, 44].
In other tissues, both Dio2 and Dio3 exhibit mild topronounced circadian profiles of expression in a mannerthat is both tissue- and sex-specific. We also observeda moderate but consistent circadian variation in theexpression of both T3 receptors across several tissues,
indicating that, barring other determinants of T3 action,
T3 receptors may also significantly contribute to circadian patterns of T3 action. Overall, these results in wild
type mice further underscore the idea that circadian patterns of T3 action are tailored to the needs of the particular tissue and are also dependent on sex.
In mice with DIO3 deficiency, we observed numerous
rhythmic disturbances. The moderate diurnal differences
in serum T4 are not present in Dio3-/- males or females.
This is not surprising as these animals show severe alterations in the programming and regulation of the hypothalamic-pituitary-thyroid axis [22, 45]. Most striking is
how DIO3 deficiency fully erases the pronounced circadian pattern of serum corticosterone in females, but not
in males. This sexually dimorphic phenotype is consistent with our recent observation of serum corticosterone
being affected in females–but not males- with DIO3 deficiency specifically in POMC-expressing cells [23], andsuggests a role for Dio3 in the physiology and circadianWu et al. BMC Molecular and Cell Biology(2024) 25:11

activity of the hypothalamic-pituitary-adrenal axis. In
this regard, the flattened time curve of serum corticosterone in Dio3-/- females may be partly explained by themarked reduction in the expression of corticosteroneproducing Cyp11b1 in the adrenal of females, precisely at
ZT12, when the hormone peaks in serum. This observation also suggests an important role for Dio3 in the regulation of corticosteroid synthesis.
DIO3 deficiency minimally affects the circadian expression of clock genes in the peripheral tissues studied. In
the hypothalamus, loss of DIO3 function does not markedly affects the overall circadian profiles of clock genes,
but the expression of a few of them show consistent andsignificant changes at ZT12 in males and ZT18 and ZT0in females, suggesting a mild influence in the regulationof the central clock. In this regard, future studies may
address a more specific role for Dio3 in the suprachiasmatic nucleus.
However, DIO3 deficiency exerts a more substantial
effect on the circadian patterns of expression of genes
regulating T3 action and regulated by T3, as well as
genes of importance to the physiology of metabolic tissues. Loss of DIO3 function generally increased hypothalamic expression of Dio3 mRNA at different ZTs inboth sexes, as Dio3 itself is a T3 target. However, there
is a circadian dependence on the existence and extent ofthat dysregulation. Dio2 mRNA expression is affected in
the hypothalamus at particular ZTs and in a sex dimorphic manner, and so is the expression of target genes Hrand Klf9. This suggests the disruption of hypothalamic
T3 action caused by DIO3 deficiency is highly dependent
on the particular ZT. A comparable conclusion can be
drawn from hepatic data, showing dysregulation of thecircadian patterns of Thrb expression (more marked inmales) and Dio1 expression (more marked in females).
Overall, the circadian-specific effects of DIO3 deficiency
on the expression patterns of T3 regulated genes suggestthat the enzymatic function of DIO3 also follows a circadian pattern in the tissues studies, as we have previously
shown in the adrenal gland [16].
In male adipose tissues, the impact of DIO3 deficiency
on circadian patterns is more pronounced. In male BAT,
the time curves of both Dio2 and Dio3 exhibits severe
disruption, which is similar to that observed for Pcg1a,a critical gene in BAT differentiation. These disruptions are associated with ZT-specific abnormalities inthe expression of important genes for the physiology of
the tissue. Based on analysis using circadian software
CircaCompare, Dio3-/- abolished circadian rhythm onlipid metabolism genes, but not on clock genes. It suggests Dio3 plays a role in mediating function between
clock genes and lipid metabolism genes. Similarly, WAT
shows marked and sex-specific disruptions in the time
curves of Dio2 and Dio3 expression. Importantly, this isPage 12 of 14

associated with major disruptions in the time curve ofgenes related to adiposity such as Lep and Mest, suggesting that highly relevant processes for white adipose tissue circadian physiology are severely disrupted by loss of
DIO3 function.
It remains uncertain what mechanisms are driving thecircadian regulation of Dio2 and Dio3 in such a sex- andtissue-specific manner. Pathways downstream of clock
genes may be playing a role. For example Bmal1 has beenshown to control the regulation of cone function of theretina by thyroid hormones [46]. Another possibility may
involve circadian glucocorticoid action, as glucocorticoids have been shown to regulate Dio2 [47, 48] and Dio3[49] in several cell culture and animal models [50–52],
with some exceptions in which no effect was observed[53]. Additional research will be required in this regard.

Conclusion
Genes involved in the regulation of thyroid hormone
action exhibit sex- and tissue-specific circadian patterns of expression. A deficiency in one of them, Dio3,
although not significantly affecting the expression ofclock genes, leads to sexually-dimorphic alterations in
the circadian profiles of corticosterone and liver andadipose tissue gene expression, impacting determinantsof thyroid hormone action and critical markers of tissue
physiology (Fig. 10). Our studies underscore the potential
importance of circadian rhythmicity in the local regulation of thyroid hormone action and the role of Dio3 inthis process, an underappreciated factor that may be ofsignificance to the metabolic pathophysiology of target
tissues.

Wu et al. BMC Molecular and Cell Biology(2024) 25:11

Page 13 of 14

Fig. 10 Summary of the circadian disruptions (highlighted in red arrows and boxes) caused by DIO3 deficiency. Dio3-/- flattened or completely erased
the circadian rhythm of gene expression in different tissues by T3 effects through the hypothalamic-pituitary-adrenal (HPA) axis without disrupting thecircadian rhythm of clock genes in the specific mouse tissue

Supplementary Information

Received: 19 December 2023 / Accepted: 15 March 2024

The online version contains supplementary material available at https://doi.
org/10.1186/s12860-024-00508-6.
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Acknowledgements
We are grateful to Victoria DeMambro for assistance with metabolic cages
data retrieval. This work utilized the Molecular Phenotyping and the
Physiology Cores at MaineHealth Institute for Research. These core facilities aresupported by grants P30GM106391, U54GM115516, and P20GM121301 fromthe National Institute of General Medical Sciences.
Author contributions
Conceptualization (ZW and AH), investigation and formal analyses (ZW and
MEM), funding acquisition (AH), visualization (ZW, AH), writing draft (ZW),
writing and editing (ZW, MEM, AH).
Funding
This research was funded by the National Institute of Mental Health, grant
number MH096050, and by the National Institute of Diabetes, Digestive andKidney Diseases, grant number DK095908.
Data availability
All data generated or analyzed during this study are included in this publishedarticle and its supplementary information files.

Declarations
Ethics approval and consent to participate
All animal procedures and this study were approved by the MaineHealthInstitute for Research Institutional Animal Care and Use Committee (IACUC).
All methods were performed in accordance with the Guide for the Care andUse of Laboratory Animals of USA.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.

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