Sexual dimorphism occurs in many fundamental metabolic processes, which likely influence the development of metabolic diseases. Understanding the sex-specific factors and pathways that promote or mitigate disease may lead to a better understanding of disease pathogenesis and useful interventions. The present results illustrate the complex interplay between several major classes of sex-specific factors that cause sexual dimorphism in obesity, and highlight the utility of the FCG model for investigating such interactions. For the first time, we demonstrate that sex chromosome complement, independent of gonadal sex, has substantial effects on body weight and adiposity in adult mice on a chow diet, and on the rate of weight gain in mice fed a high fat diet. We found that the increased adiposity observed in XX mice is attributable to the presence of two X chromosomes rather than to the lack of a Y chromosome. These results focus attention of future studies on a specific set of X chromosome genes that exhibit altered expression in metabolic tissues of XX compared to XY animals because of escape from X chromosome inactivation or sex chromosome-specific imprinting.
The role of sex hormones in the determination of body weight and adiposity has been documented in many studies in humans and rodent models. For example, gonadally intact male mice have higher body weight, and exhibit more pronounced diet-induced weight gain, than females; this sex difference is reversed partially or completely by ovariectomy of female mice 
. In humans, the loss of estrogens with menopause is associated with deposition of visceral body fat, and this effect can be ameliorated to some extent by hormone replacement therapy 
, . Modulating testosterone levels also affects adipose tissue storage in healthy young men, with testosterone levels inversely correlated with adipose tissue mass 
. Thus, it is clear that gonadal hormones play a strong role in determining sex differences in adiposity in mice and humans. However, few models have allowed the interrogation of potential genetic effects underlying sex differences independent of gonadal hormones.
In our characterization of the FCG mice, body weight and/or metabolic traits were influenced by all three of the major classes of sex-biasing factors: activational (acute) hormonal effects, long-lasting (organizational) hormonal effects, and sex chromosome effects 
. Several traits were influenced by interactions between two or more of these factors. At 75 days of age, gonadal males weighed 25–28% more than gonadal females, irrespective of their sex chromosome complement, suggesting that the sex difference is caused by gonadal secretions. That conclusion was confirmed because the sex difference disappeared by one month after gonadectomy. However, further analysis of the FCG model revealed that attributing sex differences in body weight solely to gonadal hormones would be a significant oversimplification. Prior to gonadectomy, XX mice weighed 6–9% more than XY mice, in both gonadal males and females. The XX vs. XY difference became dramatically larger after gonadectomy, with XX mice having up to 2-fold greater adiposity than XY mice. Layered on top of this was an effect of Sry
(likely mediated by long-lasting effects of the original gonadal hormones), as without their gonads, gonadal female XX mice lacking Sry
had higher body weight, fat pad mass, and plasma leptin levels than gonadal male XX mice possessing Sry
. The results indicate that although sex chromosome effects act in both intact and gonadectomized mice, gonadal hormones blunt the influence of sex chromosome complement, suggesting that the hormones may have different effects depending on the chromosomal sex of cells. Thus, understanding how males and females differ from one another is not simply a matter of studying an apparently dominant factor that causes the sex difference, but requires disentangling the interactive effects of several sex-biasing factors.
The increased body weight of XX mice was preceded by increased food intake compared to XY mice; interestingly, this was restricted to the light portion of the diurnal cycle (see ). After differences in adiposity were established between XX and XY mice, food intake was not distinguishable, but leptin levels were elevated in XX mice, suggesting relative leptin resistance in the XX mice. Since there were no detectable compensatory changes in energy expenditure or physical activity in XX mice, this increase in food intake likely contributes to the increased body weight. The increased consumption of carbohydrates during the light period was reflected in slightly elevated RQ during the same period. This difference was evident even before the GDX XX mice had increased body weight. A trend toward increased food intake during the light period continued after the XX mice were substantially heavier (at 10 months post-GDX), although it was no longer statistically significant. A recent study has shown that mouse food intake during the light period of the circadian cycle leads to greater weight gain than equivalent intake during the dark period, when mice typically consume the majority of their calories 
. Many other studies have provided evidence that energy balance is tightly integrated with the circadian clock and that disruption of this cycle has detrimental effects on many aspects of metabolism 
. Thus, a focus of future studies in the FCG model will be the investigation of whether sex chromosome complement influences regulation of the circadian clock and/or networks for food consumption and satiety.
Sex chromosome complement had a key effect on the response to a high fat diet, with XX mice having an almost immediate divergence in weight gain from XY mice. An interesting finding was the greater expansion in the subcutaneous fat depot in XX mice, and greater increase in the gonadal fat depot in XY mice. It has been shown that women store a greater percentage of dietary fatty acids in subcutaneous adipose tissue than men 
. Our observations in mice raise the possibility that sex chromosome complement may be a factor in determining the propensity to store fat in various anatomical depots.
The high fat diet also led to the development of more pronounced metabolic dysregulation in XX mice, particularly fatty liver. Non-alcoholic fatty liver disease affects up to one-third of American adults, usually in association with obesity and insulin resistance 
. The occurrence of fatty liver disease and its progression to cirrhosis, end-stage liver disease and hepatocellular carcinoma are influenced by many factors, including genetic factors. Our studies reveal that XX sex chromosome complement is one genetic factor that promotes development of fatty liver in mice. It is likely that the fatty liver in XX mice fed the high fat diet was influenced by risk factors such as increased adiposity and hyperinsulinemia. Interestingly, however, fatty liver did not parallel circulating triglyceride and fatty acid levels, which were more influenced by gonadal sex (likely influenced by organizational effects of gonadal hormones) rather than sex chromosome complement. In future studies, it will be interesting to determine whether sex chromosome complement also influences the propensity for progression of steatotic livers to cirrhosis, the basis of which is currently not understood.
The sex chromosome effects reported here indicate that inherent sex differences in expression of X chromosome genes, stemming from the difference in number or parental imprint of X genes in XX vs. XY mice, contribute to sex differences in adiposity and metabolic disease. The sex chromosome effects are unlike typical linkage of genes to phenotype, because they are not caused by differences in the genetic sequence of the X chromosome, which was identical in all mice studied. Because X-inactivation effectively reduces the inherent bias toward higher expression of X genes in XX mice relative to XY mice, prime candidates for the genes causing the adiposity are those that escape X inactivation, or those that receive a parental imprint, leading to differential expression in XX compared to XY mice 
A significant proportion of X chromosome genes (15–25%) are thought to escape X chromosome inactivation in humans 
, and most of the genes escaping X inactivation in mice also escape in humans 
. We tested expression levels of candidate genes that are known to escape inactivation in both mouse and human (Eif2s3x
) or have a Y paralogue with some evidence for higher expression in XX than XY mice and humans (Usp9x
. Each of these genes was expressed at higher levels in XX than XY gonadal fat in gonadectomized mice, providing evidence that these genes escape inactivation in a metabolic tissue. Thus, these genes are candidates for those causing the XX–XY differences in physiology and adiposity reported here. Alternatively, differential expression of X chromosomes escapee genes could occur secondarily to differences in adiposity between XX and XY mice, in which case they may be downstream players in the observed metabolic differences. In addition to sex chromosome genes, autosomal genes that are differentially expressed in response to X chromosome gene dosage may contribute to the observed metabolic differences between XX and XY mice. It is known, for example, that the dysregulation of genes involved in mitochondrial metabolism and protein translation occurs in tissues of XX compared to XO mice, but the metabolic consequences are not known 
A reasonable question is whether these studies in the mouse have relevance to obesity in humans. Unusual numbers of sex chromosomes in human conditions such as Klinefelter (XXY) and Turner (XO) syndromes are associated with metabolic disease and/or adiposity 
. However, in these diseases, endocrine abnormalities may contribute and are difficult to distinguish from the sex chromosome effects. The utility of our model is that it is genetically tractable in a way that human studies are not. Since fundamental genetic and metabolic processes are shared between mice and humans, we propose that the identification of X-linked genes that have a large impact on obesity in the mouse could lead to the discovery of novel mechanisms that impact obesity in humans. The increasing longevity of the human population means that the hypogonadal period may extend for up to half of a persons' lifetime, and the inherent genetic sex differences uncovered here may have important ramifications. Furthermore, since the gene content of the X chromosome is conserved in mouse and human, and several of the same genes escape inactivation in both species, there is hope that characterizing the action of X gene(s) in mouse will advance our understanding of human metabolic disease.