The number of x chromosomes causes sex differences in adiposity in mice

Abstract

Sexual dimorphism in body weight, fat distribution, and metabolic disease has been attributed largely to differential effects of male and female gonadal hormones. Here, we report that the number of X chromosomes within cells also contributes to these sex differences. We employed a unique mouse model, known as the "four core genotypes," to distinguish between effects of gonadal sex (testes or ovaries) and sex chromosomes (XX or XY). With this model, we produced gonadal male and female mice carrying XX or XY sex chromosome complements. Mice were gonadectomized to remove the acute effects of gonadal hormones and to uncover effects of sex chromosome complement on obesity. Mice with XX sex chromosomes (relative to XY), regardless of their type of gonad, had up to 2-fold increased adiposity and greater food intake during daylight hours, when mice are normally inactive. Mice with two X chromosomes also had accelerated weight gain on a high fat diet and developed fatty liver and elevated lipid and insulin levels. Further genetic studies with mice carrying XO and XXY chromosome complements revealed that the differences between XX and XY mice are attributable to dosage of the X chromosome, rather than effects of the Y chromosome. A subset of genes that escape X chromosome inactivation exhibited higher expression levels in adipose tissue and liver of XX compared to XY mice, and may contribute to the sex differences in obesity. Overall, our study is the first to identify sex chromosome complement, a factor distinguishing all male and female cells, as a cause of sex differences in obesity and metabolism.

Figure 1. Increased body weight and fat mass in XX versus XY mice on a chow diet.

(A) Body weight of four core genotype (FCG) mice at day 21 and day 45, prior to gonadectomy (GDX), and at 10 months after GDX. F, gonadal female; M, gonadal male. Values represent the mean ± SEM for the number of animals indicated in each bar. Significant comparisons for sex chromosome complement and for gonadal sex are denoted by brackets. A significant interaction of sex chromosome complement and gonadal sex is denoted by “Int.”. The p values are as described at the end of the legend. (B) Body weight curve for FCG mice from the point of gonadectomy through 10 months following gonadectomy. Values represent mean ± SEM. Values are significantly different between mice with XX vs. XY genotypes beginning at week 7 following GDX and beyond that. (C) Body composition of mice in panel (B) determined by NMR in FCG mice at 10 months after GDX. (D) Fat pad mass in mice from panel (B) at 10 months following GDX expressed as absolute mass (grams) or relative to kidney weight, which is invariant among the genotypes. (E) Plasma leptin levels and leptin mRNA levels in inguinal adipose tissue. *, p<0.05; **, p<0.01; ***, p<0.001; †, p<0.0001; ‡, p<0.000001.

Figure 2. Altered food intake and RQ in XX versus XY mice.

Mice were individually housed in metabolic cages to assess food take and energy balance parameters continually throughout the diurnal cycle. (A, B) Food intake determined at 4 weeks or 10 months following GDX. Values represent the mean ± SEM food intake summed over two dark or two light periods. Data shown represent raw values; normalization to body weight or to lean body mass gave the same outcome. (C) Food intake patterns throughout 3 nights and 2 days. At left, lines represent mean values for XX (n = 8) and XY (n = 10) mice; at right, lines represent mean values for gonadal females (n = 9) and gonadal males (n = 9). (D, E) Mean Respiratory Quotient (RQ) ± SEM for light and dark periods determined at 4 weeks or 10 months following GDX. At right, the change in RQ between dark and light periods (Δ RQ) is shown. *, p<0.05; **, p<0.01; ***, p<0.001; ‡, p<0.000001.

Figure 3. Enhanced weight gain and fat mass in XX compared to XY mice fed a high fat-high carbohydrate diet.

(A) FCG mice were fed a high fat-high carbohydrate diet beginning at 4 weeks following GDX (week 0). XX mice first exhibited significantly higher body weight beginning at 3 days on the diet (arrow), and throughout the remainder of the study. (B) Body composition was determined by NMR at 16 weeks of the high fat-high carbohydrate diet. Fat and lean mass are shown as absolute mass and as percent of total body weight (BW). (C) Plasma leptin levels were higher in XX mice than XY mice at the end of the 16 week high fat diet period, and higher in gonadal females than gonadal males. (D) Tissue weights were determined by excision after 16 weeks on the high fat-high carbohydrate diet. Kidney weight did not differ among the four genotypes. Inguinal and gonadal fat pads are shown as absolute weights and normalized to kidney weight. Values shown for all bars represent mean ± SEM for the number of each genotype indicated. *, p<0.05; **, p<0.01; ***, p<0.001; †, p<0.0001; ‡, p<0.000001.

Figure 4. Diet-induced fatty liver and impaired glucose homeostasis are more pronounced in XX than XY mice.

FCG mice were fed a high fat-high carbohydrate diet for 16 weeks. All values represent mean ± SEM. (A) XX mice had increased liver mass and hepatic triglyceride levels compared to XY mice. (B) Hematoxylin and eosin staining of liver sections shows hepatic lipid droplet accumulation in XX mice. In the lower panel, thin layer chromatography demonstrates increased triglyceride levels in liver of XX mice relative to XY mice. (C) Fasting glucose and insulin levels, and homeostatic model assessment (HOMA) were determined. XX mice had significantly higher insulin levels than XY mice. (D) Fasting plasma triglyceride (TG) and free fatty acid (FFA) levels were higher in gonadal males than in gonadal females. (E) Increased triglyceride accumulation in XX liver is associated with increased peroxisome proliferator-activated receptor γ (Pparg) and diacylglycerol acyltransferase 1 (Dgat1) mRNA levels. (F) Increased expression levels for fatty acid oxidation genes acyl CoA oxidase 1 (Aox1) and carnitine palmitoyltransferase 1α (Cpt1a) in liver of XX mice. (G) Increased expression of Aox1 in skeletal muscle of gonadal male mice. *, p<0.05; **, p<0.01; ***, p<0.001; †, p<0.0001.

Figure 5. The number of X chromosomes determines differences in body weight and adiposity.

(A) Body weight following gonadectomy at day 75 of mice having the indicated sex chromosome complements fed a chow diet. The Y* chromosome has been described , and the near equivalent genotype in terms of X and Y chromosome complement is shown. Two groups of mice with two X chromosomes had higher body weight, relative to mice with one X chromosome, beginning at 8 weeks after GDX and thereafter (p<0.000001). Mice with a Y chromosome did not differ from mice lacking a Y chromosome. (B) Body weight and body composition determined by NMR in mice shown in panel (A) at 9 months after GDX. Values shown for all bars represent mean ± SEM for the number of each genotype indicated. **, p<0.01; ***, p<0.001; †, p<0.0001; ‡, p<0.000001.

Figure 6. Differential gene expression in liver and fat tissues of X chromosome genes that escape inactivation.

Genes previously shown to escape X chromosome inactivation (see text) were assessed for expression levels by quantitative PCR in liver and adipose tissue (subcutaneous inguinal and gonadal depots) of gonadectomized, chow fed FCG mice shown in Figure 1B (10 months post-GDX). (A) Statistical differences in gene expression levels among the FCG genotypes for genes escaping X chromosome inactivation. For each gene, the p value for differences between XX and XY, and female (F) vs. male (M), are shown. Several escapees exhibit increased expression in liver and/or adipose tissues of XX compared to XY mice; a few genes also exhibit differences between levels in gonadal females and gonadal males. Mid1 shows a unique pattern, with lower expression levels in XX compared to XY tissues. The full name for Rik is 2610029G23Rik. NS, not significantly different. (B–D) mRNA levels are shown for liver and gonadal fat tissue of FCG mice for Eif2s3x, Kdm6a, and Ddx3x. Values shown for all bars represent mean ± SEM for the number of each genotype indicated. **, p<0.01; ‡, p<0.000001.