Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity

Results

Body and Fat Pad Masses.

Adult female ArKO mice were significantly heavier and had significantly larger gonadal and infra-renal fat pads than WT littermates from 3 months of age onward (Fig. 1 A and C and Table 1). Male ArKO mice also had significantly heavier gonadal fat pads than WT males from 3 months of age and heavier infrarenal fat pads from 4 months of age (Fig. 1 B and D and Table 1). However, their body weight failed to show a significant increase until 1 year of age.

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Figure 1

Body mass and gonadal fat pad mass for ArKO (filled bars) and WT (empty bars) mice. (A) Female body mass. (B) Male body mass. (C) Female gonadal fat pad mass (two pads). (D) Male gonadal fat pad (pad) mass. Minimum six animals per group. *, P ≤ 0.05.

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Table 1

Infrarenal fat pad masses

Adipocyte Volume.

Adipose tissue accretion may be due to an increase in the number of adipocytes or an increase in the volume of individual adipocytes, or both. Visual assessment of gonadal fat tissue from 1-year-old male animals indicated that adipocyte size was larger in ArKO animals than in WT animals (Fig. 2). To evaluate adipocyte volume, stereological analyses were conducted. As shown in Fig. 3, adipocyte volume is unchanged over time in WT control animals, and cell volume appears to be very similar between WT males and females. At 3 months of age, adipocyte volume was the same between knockout and control animals for both genders. By 4 months of age, however, there was a significant increase in the volume of individual adipocytes in the gonadal fat pads of male and female ArKO mice when compared to WT mice. This increase in cell volume was maintained through 1 year of age.

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Figure 2

Gonadal fat pads from 1-year-old male mice were fixed, sectioned, and stained with hematoxylin and eosin. (A) WT. (B) ArKO. (Bar represents 50 μm.)

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Figure 3

Adipocyte volume data for female (A) and male (B) mice. Empty bars, WT; filled bars, ArKO. Minimum five animals per group. *, P ≤ 0.05.

Fatty Liver.

General inspection of internal organs at necropsy revealed that in 1-year-old ArKO animals the liver was often larger and paler in color than that of WT littermates. After fixation, sectioning, and staining, the livers of 3-month-old and 1-year-old ArKO animals were observed to have a greater accumulation of lipid droplets than WT control animals (Fig. 4).

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Figure 4

(A and B) Sectioned livers of 3-month-old (A) and 1-year-old (B) WT males. (C and D) Sectioned livers of 3-month-old (C) and 1-year-old (D) ArKO males. (Bar represents 50 μm.)

Discussion

The present studies reinforce and extend the concept that estrogens play an important role in the regulation of adiposity as a function of age in both males and females. Estrogens have important roles in adipose tissue, within the central nervous system, in bone, and in other extragonadal sites (13). The present study has shown that ArKO mice of both sexes with estrogen insufficiency develop a progressive increase in adiposity due to accumulation of intraabdominal fat. This was associated with an increase in adipocyte volume, hyperleptinemia, hyperinsulinemia, and hypercholesterolemia. However, unlike many other rodent models of obesity, excess body fat in the estrogen-insufficient ArKO mice was not due to hyperphagia or reduced resting energy expenditure, but was associated with decreased lean mass and reduced spontaneous physical activity. The obese phenotype is likely to be due to estrogen insufficiency rather than testosterone excess because 17β-estradiol treatment normalized body fat content in the ArKO mice, and the obese phenotype is similar in male and female ArKO mice despite their greatly different testosterone levels. Furthermore, a similar phenotype of fat accumulation is observed in estrogen-deficient ovariectomized rats, which cannot synthesize androgens (14).

An increase in adiposity and a decrease in lean mass in the ArKO mice suggests that estrogen affects nutrient partitioning. There are several possible biochemical mechanisms by which estrogen may have this effect. For example, oophorectomy is known to impair muscle glucose uptake and storage (15) in rats, which could decrease lean body mass. This decrease in glucose utilization by muscle would make more glucose carbon available for lipogenesis and could promote fat deposition. Muscle is responsible for most postprandial glucose uptake, so this decrease in muscle glucose uptake would also be expected to reduce whole body glucose oxidation and induce insulin resistance, as we have observed in the older ArKO mice. Alternatively, altered nutrient partitioning in the estrogen-insufficient ArKO mice may be due to impaired uncoupling protein function. Hope et al. (16) have shown decreased UCP2 mRNA expression in skeletal muscle but not adipose tissue of ovariectomized dunnarts (Sminthopsis crassicaudata) compared with sham-operated animals. This may reduce the utilization of nutrients specifically in muscle, reducing lean body mass and increasing adipose tissue mass.

The reduced spontaneous physical activity we found in the ArKO mice may also be partly responsible for the increase in fat pad mass. It is of interest that reduced physical activity is also reported to be a feature of estrogen deficiency in both ovariectomized animals (14) and postmenopausal women (17). Reduced physical activity in the ArKO mice may be secondary to the increase in fat mass and decrease in lean body mass seen in these mice. However, it is also possible that this inactivity is caused by an effect of estrogen insufficiency on the function of the central nervous system in these mice.

The ArKO mice had circulating leptin levels which were 2- to 3-fold higher than those of the WT mice from 4 months of age. This observation is not surprising, given the established association between obesity and increased production of leptin in most human subjects (18) and most animal models of obesity (19–22). Leptin regulates body fat predominantly by decreasing food intake (23). The fact that food intake is reduced in our ArKO mice suggests that these mice are responding normally to leptin. This fits with data showing that sensitivity to an intracerebroventricular dose of leptin is normal in obese ovariectomized mice, which have 59% lower estrogen levels than intact mice (24).

In older ArKO animals, the adiposity was associated with hypercholesterolemia and, at least in males, hypertriglyceridemia. The hypercholesterolemia was reflected in elevated HDL. Two aromatase-deficient men with mutations in the human CYP19 gene have also been reported to have altered circulating lipid profiles (4, 5). Blood serum levels of cholesterol, low density lipoprotein (LDL), and triglycerides were elevated in both men, but the concentration of HDL was decreased. Treatment with estradiol restored these parameters closer to normal limits. The different HDL response in aromatase-deficient mice and humans is likely due to the fact that the dominant lipoprotein involved in delivery of cholesterol to extrahepatic tissues and the liver (25, 26) is HDL in rodents, whereas LDL serves this role in humans. Hence it is not surprising that elevated serum cholesterol in mice is reflective, at least in part, of elevated HDL. The action of estrogens to lower circulating cholesterol in the form of LDL levels in humans has been implicated in their cardioprotective action (27). Nevertheless, oral estrogen increases serum triglyceride levels (27). This increase has been attributed to the “first pass” effect of oral administration exposing the liver to high concentrations of estrogen, because it is not seen with parenteral administration (28). Thus, the aromatase-deficiency phenotype and the results of parenteral administration would suggest that estrogens at circulating physiological concentrations do not increase serum triglycerides, and may in fact decrease them.

The observation that ArKO mice develop a fatty liver phenotype may be related to the observed hypercholesterolemia. One of the ways in which estrogen is believed to lower circulating cholesterol is by increasing the level of LDL receptor expression in the liver, thus enhancing LDL uptake (29). Moreover, mice deficient in the oxysterol receptor LXR (LXR^−/−) fail to induce transcription of cholesterol 7α-hydroxylase (Cyp7a), the rate-limiting enzyme in bile acid synthesis, and accumulate cholesterol in the liver and have marked hypercholesterolemia (30). It has been reported that estrogen and progesterone increase the activity of cholesterol 7α-hydroxylase (31), so it is conceivable that both the fatty liver and hypercholestrolemia in the ArKO mice are due to a reduction in bile acid synthesis secondary to estrogen deficiency. Concomitant with a possible perturbation in this pathway, Nemoto et al. (32) report impairment in hepatocellular fatty acid β-oxidation in an aromatase-deficient mouse model they have independently generated. Analyses revealed a decrease in both mRNA expression and activities of specific enzymes required in fatty acid β-oxidation. Evidently, estrogens play multiple roles in lipid homeostasis, and are crucially involved not only in adipose depot accretion, but also in lipid turnover in the liver.

Associated with both increasing age and fat accumulation was a 4-fold increase in circulating levels of insulin, glucose levels remaining unchanged. Normoglycemia at the expense of hyperinsulinemia is suggestive of insulin resistance in 1-year-old ArKO mice. Elevated insulin levels are also found in humans with aromatase deficiency (3–5). Insulin resistance is a characteristic of obesity. The mechanism underlying this is unclear, but it appears to involve elevated free fatty acids and tumor necrosis factor α (33, 34).

Evidence that estrogen action plays an important role in body fat deposition is provided not only by these studies on the ArKO mouse, but also by two other rodent models of estrogen deprivation. Ovariectomized rats show an increase in body mass and fat deposition (14), as do α-estrogen receptor knockout (αERKO) mice (35). β-Estrogen receptor knockout (βERKO) mice, on the contrary, do not accumulate excess adipose tissue (36). This observation suggests that estrogen action, in terms of accumulation of fat, is mediated via ERα rather than ERβ, which is consistent with reports by ourselves and others that ERα, but not ERβ, is present in adipose tissue (ref. 36, ‡‡).

At present it is not known whether the increase in abdominal adiposity resulting from estrogen deficiency reflects direct actions of estrogens on the adipose depots or central actions or both. Estrogen is known to have direct effects on adipose tissue, e.g., oophorectomy decreases lipolysis in adipose tissue (37) of rats and estradiol treatment lowers fatty acid synthesis and increases lipolysis in fat cells (38). However, estrogen may also be involved in the central regulation of body weight, e.g., estrogen deficiency is known to increase hypothalamic neuropeptide Y expression (39) and decrease hypothalamic corticotropin-releasing factor immunoreactivity (40). Alternatively, estrogen may centrally regulate body weight by enhancing glucose uptake into the brain (41), a process that may facilitate recognition of the fed state. The fact that estrogen receptors are expressed within the central nervous system makes it possible that obesity in the ArKO and αERKO mice (35) is due, at least in part, to impaired signaling of estrogen through the α-estrogen receptor found in the hypothalamus of the brain. Further investigations are needed to unravel the cellular and biochemical mechanisms underlying the physiological phenomena reported here.