Mice lacking DIO3 exhibit sex-specific alterations in circadian patterns of corticosterone and gene expression in metabolic tissues

Abstract

Disruption of circadian rhythms is associated with neurological, endocrine and metabolic pathologies. We have recently 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 the physiological 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 genes that determine tissue thyroid hormone action and physiology. We also observed circadian-specific changes in serum 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 the metabolic phenotypes of DIO3-deficient mice.

Keywords: Adipose tissue; Circadian rhythm; Clock genes; Corticosterone; Hypothalamus; Liver; Thyroid hormone action; Type 2 deiodinase; Type 3 deiodinase.

Fig. 1

Circadian parameters of running wheel data of adult Dio3-/- mice. Typical 48 h of data was analyzed using CircaCompare, according to Parsons et al., 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 and T4 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 mice

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 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

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

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 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

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 per experimental group and ZT. *P < 0.05, **P < 0.01, as determined by two-way ANOVA and Tukey’s post hoc test

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

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

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

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 the circadian rhythm of clock genes in the specific mouse tissue