The circadian clock components CRY1 and CRY2 are necessary to sustain sex dimorphism in mouse liver metabolism

Abstract

In mammals, males and females exhibit anatomical, hormonal, and metabolic differences. A major example of such sex dimorphism in mouse involves hepatic drug metabolism, which is also a noticeable target of circadian timekeeping. However, whether the circadian clock itself contributes to sex-biased metabolism has remained unknown, although several daily output parameters differ between sexes in a number of species, including humans. Here we show that dimorphic liver metabolism is altered when the circadian regulators Cryptochromes, Cry1 and Cry2, are inactivated. Indeed, double mutant Cry1(-/-) Cry2(-/-) male mice that lack a functional circadian clock express a number of sex-specific liver products, including several cytochrome P450 enzymes, at levels close to those measured in females. In addition, body growth of Cry-deficient mice is impaired, also in a sex-biased manner, and this phenotype goes along with an altered pattern of circulating growth hormone (GH) in mutant males, specifically. It is noteworthy that hormonal injections able to mimic male GH pulses reversed the feminized gene expression profile in the liver of Cry1(-/-) Cry2(-/-) males. Altogether, our observations suggest that the 24-h clock paces the dimorphic ultradian pulsatility of GH that is responsible for sex-dependent liver activity. We thus conclude that circadian timing, sex dimorphism, and liver metabolism are finely interconnected.

FIGURE 1.

The sex dimorphism in gene expression is reduced in the liver of Cry^–/– mice. Livers from control and Cry^–/– mice were collected every 4 h. Relative RNA levels, measured by quantitative PCR, are shown for control wild type (WT, closed symbols, solid lines) and Cry^–/– (open symbols, dotted lines) males (squares) and females (circles), respectively. Data from ZT0 are replotted at ZT24 to improve readability. RNA levels are graphed as means ± S.E., n = 3–4 for each series. For all genes, the global statistical significance is p < 0.005 between control males and females and between males of both genotypes (two-way ANOVA). Insets show the average ratio of expression over 24 h between male and female livers (M/F ratio) in wild type (solid bars) and Cry^–/– mice (open bars). **, p < 0.01 and *, p < 0.05 as compared with control males, Mann-Whitney U test.

FIGURE 2.

Sex differences in liver microsomal testosterone hydroxylase activities are reduced in Cry^–/– mice. Testosterone hydroxylase activities were determined in liver microsomes prepared from wild type (WT, solid bars) and Cry^–/– mice (open bars). Specific activities are shown for male-predominant 16α-hydroxylase (A), female-predominant 6α-hydroxylase and 7α-hydroxylase (B), and 15β-hydroxylase (C). D, activity ratios between male and female microsomes are plotted on a log scale. Note that this ratio tends to 1 (horizontal dashed line) for microsomal activities measured from Cry^–/– mice in A and B, whereas it remains constant for 15β-hydroxylase activity. **, p < 0.01 and *, p < 0.05 as compared with control males, ++, p < 0.01 and +, p < 0.05 as compared with control females and ##, p < 0.01 and #, p < 0.05 as compared with Cry^–/– males; symbols are omitted when differences are not significant (two-way ANOVA; n = 5 animals in each condition, means ± S.E.).

FIGURE 3.

The GH-dependent sex dimorphism is suppressed in Cry^–/–mice. A, body weight of animals obtained from Cry1^+/–/Cry2^+/– intercrosses. Wild type (black solid line), Cry^–/– (dotted line) and littermates bearing at least one wild type Cry allele (gray solid lines), males (upper panel) and females (lower panel), were weighted weekly from 1 to 8 weeks of age and at 12 weeks. Data are graphed as means ± S.E. ***, p < 0.005, **, p < 0.01, and ns, not significant, for control versus Cry^–/– mice, Mann-Whitney U test. B, male/female body weight ratios for wild type (WT, solid line) and Cry^–/– (dotted line) mice. Note that ratio values are close to 1 in Cry^–/– mice, indicating a loss of sexually dimorphic growth rates. C, MUPs accumulation. A representative example of MUPs stained with Coomassie Blue after SDS-polyacrylamide gel electrophoresis (upper panel) and pooled analysis of MUPs content in urine of wild type (solid bars, n = 9 males and n = 14 females) and Cry^–/– (open bars, n = 15 males and n = 12 females) is shown. ***, p < 0.005 and **, p < 0.01 as compared with wild type males (two-way ANOVA). D, Mup1 expression level in the liver of mice used in Fig. 1. The global statistical significance by two-way ANOVA is p < 0.001 between control males and females and between males of both genotypes.

FIGURE 4.

Circulating GH levels are altered in Cry^–/– males. A, box plots of circulating GH levels measured in trunk blood randomly collected between ZT2 and ZT8 from wild type (WT, solid boxes with median in white, n = 30 males and 30 females) and Cry^–/– mice (open boxes with median in black, n = 20 males and 30 females) of each sex. Individual values outside 90% of the population distributions are plotted as black circles. The median values of the four groups are significantly different (p < 0.05, Kruskal-Wallis test). **, p < 0.01 between wild type and Cry^–/– males (Dunn's multiple comparison test). B, the total amount of GH was quantified from whole pituitary glands of Cry^–/– (open circles) and control (solid circles) males and females. The inactivation of Cry genes has no effect on average GH levels (p = 0.20 and p = 0.15 for control versus Cry^–/– mice in males and females, respectively, Mann-Whitney U test). C, expression levels of genes involved in GH signaling were measured in the liver from mice used in Fig. 1. RNA levels are graphed as means ± S.E., n = 3–4 for each series. The global statistical significance by two-way ANOVA is p < 0.001 and p < 0.05 between males of both genotypes for Ghr and Igfbp3, respectively. No significant alteration was found for Igf1 in Cry^–/– males as compared with controls. Ghr, GH receptor.

FIGURE 5.

Aged male mice have a feminized liver gene pattern. Livers of 8-week-old (solid bars) and 2-year-old (open bars) C57BL/6 male mice were collected between ZT2 and ZT4. Sex-specific liver genes show a feminization of their expression level in aged males. Relative RNA levels are graphed as means ± S.E., n = 5 animals in each condition. **, p < 0.01 and *, p < 0.05, Mann-Whitney U test.

FIGURE 6.

Patterned GH pulses reverse the feminized phenotype of Cry^–/– males. Liver gene expression was assessed at ZT4 in Cry^–/– males that received two subcutaneous injections a day, at ZT0 and ZT12 during 1 week, of saline (n = 4), 50 μg of highly purified pituitary bovine GH (bGH, n = 5), or 50 ng of octreotide (n = 5), and untreated control males (n = 5). Both hormonal treatments restored a control-like expression pattern in the liver of Cry^–/– males. **, p < 0.01 and *, p < 0.05 as compared with NaCl, Mann-Whitney U test. WT, wild type.