A Guide for the Design of Pre-clinical Studies on Sex Differences in Metabolism

Abstract

In animal models, the physiological systems involved in metabolic homeostasis exhibit a sex difference. Investigators often use male rodents because they show metabolic disease better than females. Thus, females are not used precisely because of an acknowledged sex difference that represents an opportunity to understand novel factors reducing metabolic disease more in one sex than the other. The National Institutes of Health (NIH) mandate to consider sex as a biological variable in preclinical research places new demands on investigators and peer reviewers who often lack expertise in model systems and experimental paradigms used in the study of sex differences. This Perspective discusses experimental design and interpretation in studies addressing the mechanisms of sex differences in metabolic homeostasis and disease, using animal models and cells. We also highlight current limitations in research tools and attitudes that threaten to delay progress in studies of sex differences in basic animal research.

Figure 1. Origins of sex differences

Sex differences in physiology begin during development from the combination of genetic and hormonal events and they continue after puberty. They result from the combination of the cell autonomous effect of sex chromosomes, the organizational action (masculinization) of the testicular testosterone surge in males and the activational effect of male and females sex hormones acting on their receptors after puberty. T: testosterone; AR, androgen receptor; ER, estrogen receptor; GPER, G protein coupled ER; PR, progesterone receptor.

Figure 2. Mouse models to study the role of gonadal hormones

(A) A sex-biased trait is hypothesized to be due to E2 action in females. (B) This question is addressed by performing ovariectomy (OVX) in females to suppress ovarian E2 in a three group design: gonad-intact sham operated controls, OVX with vehicle treatment and OVX with E2 replacement. If OVX abolishes the sex difference in the trait (and makes the female like the male), then E2 replacement therapy should be performed to ascertain that E2 restores the phenotype of the intact female, supporting the concept that E2 contributes to the sex difference in the trait. (C) To determine which estrogen receptor (ER) mediates E2 effect; one can use selective ERα (PPT), ERβ (DPN) or the G protein-coupled ER (G1) agonists in OVX mice followed by mice with a knockout of the target ER, globally and in a tissue-specific manner.

Figure 3. Decision tree to study sex differences

Diagram showing steps to investigate the sex-biased factors that cause a sex difference in animals. Investigators start by comparing the phenotype of the two sexes, keeping environmental conditions similar, which reveals a sex difference in a trait. The next step is to vary levels of gonadal hormones in adulthood at the time of testing using gonadectomy and replacement of hormones, to determine if gonadal hormones explain the sex difference. If these manipulations show an effect, the investigators then determines the hormone receptor that mediates the effect, and the downstream molecular pathways causing the phenotypic sex difference. If such “activational” effects of hormones do not completely explain the effect, then the investigator may test for “organizational” effects of perinatal testosterone. This is done by interfering with testosterone actions or exposing females to testosterone at that period of life. If the investigator finds an effect of hormone perinatally, which causes a sex difference later in life, the finding leads to identification of the receptors involved, their sites of action, and downstream molecular mechanisms. If both of these types of manipulations of gonadal hormones do not completely explain the sex difference, then the investigator may test for the effect of sex chromosomes in specific mouse models are appropriate (Four Core Genotypes and XY*). Even if there is no sex difference in the overt phenotype, there may exist sex differences in underlying mechanisms, which cancel each other out. This figure is adapted from (Becker et al., 2005).

Figure 4. Mouse models to study the role of X and Y chromosomes

(A) Left: The Four Core Genotypes model distinguishes effects that correlate with XX versus XY sex chromosome complement from effects that differ based on male vs. female gonads. Right: body weight gain on a high fiat diet is accelerated in XX compared to XY mice that were gonadectomized as adults. From (Chen et al., 2012). (B) Left: After the identification of differences between XX and XY mice, the XY* model is used to investigate the contribution of X versus Y chromosome copy number. Right: Gonads were removed from adult mice (time 0) and 4 weeks later the body weights converged, followed by increased weight gain in mice with two X chromosomes (XX and XXY) compared to those with one X chromosome (XO and XY). The presence of the Y chromosome does not affect body weight gain. From (Chen et al., 2012). (C) Left: The Sex Chromosome Trisomy model assesses differences related to one or two X chromosomes with a Y chromosome and different gonadal combinations. Right: Animals were examined with either intact gonads or after gonadectomy (GDX) and delivery of testosterone (T) to normalize levels across genotypes. In both conditions, presence of two X chromosomes led to increased percent body fat compared to a single X chromosome. From (Chen et al., 2013). (D). FCG studies lend themselves to analysis by 2-way ANOVA, with gonadal sex and sex chromosomes as main effects. These analyses can reveal (Left) a main effect of gonadal sex, (Middle) a main effect of chromosomal sex, or (Right) an interaction between gonadal and chromosomal sex.

Figure 5. Male and female biological systems

Sex differences in vivo result from the sum of all sex-specific influences on cellular systems including hormones, metabolites, neural inputs, etc. They define two different male and female biological systems or “sexome”. When primary cells are isolated and cultured, the sex differences in the cells’ phenotype can come from sex chromosome effects or be caused by transient (e.g., gonadal hormone levels modifying gene expression) or permanent (epigenetic modifications induced by perinatal testosterone) sex differences present in the cells’ environments prior to harvest, which are carried over into the dish.