Dietary xenosterols lead to infertility and loss of abdominal adipose tissue in sterolin-deficient mice

Abstract

The investigation of the human disease sitosterolemia (MIM 210250) has shed light not only on the pathways by which dietary sterols may traffic but also on how the mammalian body rids itself of cholesterol and defends against xenosterols. Two genes, ABCG5 and ABCG8, located at the sitosterolemia locus, each encodes a membrane-bound ABC half-transporter and constitutes a functional unit whose activity has now been shown to account for biliary and intestinal sterol excretion. Knockout mice deficient in Abcg5 or Abcg8 recapitulate many of the phenotypic features of sitosterolemia. During the course of our studies to characterize these knockout mice, we noted that these mice, raised on normal rodent chow, exhibited infertility as well as loss of abdominal fat. We show that, although sitosterolemia does not lead to any structural defects or to any overt endocrine defects, fertility could be restored if xenosterols are specifically blocked from entry and that the loss of fat is also reversed by a variety of maneuvers that limit xenosterol accumulation. These studies show that xenosterols may have a significant biological impact on normal mammalian physiology and that the Abcg5 or Abcg8 knockout mouse model may prove useful in investigating the role of xenosterols on mammalian physiology.

Fig. 1.

Gonadal histology from Abcg8 knockout mice. Abcg8 wild-type or knockout mice were raised on rodent chow. Ovaries (A and B) or testes (C and D) were obtained from adult animals (ages > 12 weeks), fixed, sectioned, and stained with H and E. (A and C) are from wild-type mice, and (B and D) are from Abcg8 knockout mice. No significant differences were discernible between wild-type and knockout mice.

Fig. 2.

Hormonal and infertility profiles in ABCG8 knockout mice. To investigate the infertility of Abcg8 knockout mice, sperm motility (A), male FSH levels (B), female prolactin levels (C), male testosterone (D), male di-hydrotestosterone (E), and female estrus cycles (F) were examined. Except for estrus cycles, no differences were noted between wild-type (open bars) and knockout mice (black bars). Estrus cycles were lengthened by at least one day, and all of the increase was in the postestrus phase (see text). Fortification of the rodent chow diet with the sterol absorption-blocking drug ezetimibe reversed this and restored fertility (see text).

Fig. 3.

Effect of meat-only diet in Abcg8 knockout mice. To exclude plant sterols from the diet to see whether fertility could be restored, we weaned female mice and placed these on the meat diet (see Methods). At the end of the experiment (after breeding challenge), plasma was examined by GC for cholesterol (A) and sitosterol (B). On the meat diet, Abcg8 knockout mice (B, middle bars, n = 5) showed significantly lower plant sterol levels compared with knockout mice fed chow (B, n = 4), and although the cholesterol levels were also decreased (A, middle bar), these did not reach statistical significance by ANOVA. Note that the plant sterols attained on the meat diet in the Abcg8 knockout mice were only 1.1 ± 0.4 mg/dl compared with wild-type mice (n = 6) fed the meat diet, 0.5 mg/dl (lower level of detection). On chow, the Abcg8 knockout mice (n = 4) had a sitosterol level of 79 ± 35 mg/dl. All of the Abcg8 knockout mice on the meat diets got pregnant, whereas none of the knockout mice on the chow diet did.

Fig. 4.

Effect of villinTg-ABCG8 gene expression in Abcg8 knockout mice on sterol profiles. Mice expressing human ABCG8, driven by the intestine-specific villin promoter, were made and bred into the Abcg8 knockout line (see Methods) and placed on chow or a high-sitosterol (HS) diet. On chow or HS diet, plasma cholesterol levels (A) were not altered by the presence of transgene (the genotypes are as indicated on the x axis). On a HS diet, plasma sitosterol levels were significantly increased in villin Tg mice (B, second bar) compared with mice raised on chow alone. However, these levels are small (1.7 ± 1.2 mg/dl, n = 4). On a chow diet, villin Tg-Abcg8 knockout mice (B, third bar) showed very mild elevations in plasma sitosterol levels (6.3 ± 1 mg/dl), and more remarkably, when placed upon a HS diet (B, last bar), showed significantly higher, but greatly attenuated levels (10.3 ± 1.2 mg/dl) compared with the chow group. Note that hepatic deficiency of Abcg8 function was not altered by the transgene expression; liver sitosterol levels were high in villinTg-Abcg8 KO mice (D), and biliary excretion of cholesterol and sitosterol remained low (E and F). Although villinTg mice on a HS diet showed increased liver cholesterol levels compared with chow, this observation was not pursued further (C).

Fig. 5.

Quantitation of regional fat depots in Abcg8 knockout mice. Fat depots as indicated were dissected, weighed, and normalized to body weights in wild-type (open bars, n = 4), knockout (black bars, n = 4), or knockout mice fed chow supplemented with ezetimibe (gray bars, n = 4 for males and n = 4 for females). Mice were ∼16 weeks old at sacrifice and were fed chow. In knockout mice, fat depots were significantly reduced in the gonadal (A and B) as well as perinephric area (C and D), whereas in knockout mice fed chow supplemented with ezetimibe, there were no differences when compared with wild-type mice raised on chow.

Fig. 6.

Growth charts for Abcg8 knockout mice. Serial weights of male (A) and female mice (B) were monitored. On chow, Abcg8 knockout mice (filled circles) do not show any significant differences compared with wild-type mice. Food intake was determined by serial food weighing and no differences (C) (males 4.30 ± 0.98 versus 4.53 ± 0.48 g/day; females 4.07 ± 1.19 versus 4.56 ± 0.50 g/day, respectively) were noted. Activity levels also seemed comparable (D) (males 9.51 ± 1.99 versus 10.47 ± 1.9 counts/min; females 13.53 ± 0.93 versus 15.46 ± 1.93 counts/min, respectively).

Fig. 7.

Adipocyte sizes are reduced in Abcg8 knockout mice and reversed by ezetimibe. (A and B) Representative histological sizes of adipocytes from fat stores from wild-type and knockout mice, respectively. Formal size quantitation is shown below: (C) wild-type, (D) Abcg8 knockout on chow, € Abcg8 knockout on chow supplemented with ezetimibe. The smaller adipocyte cell sizes are restored toward normal in Abcg8 knockout mice when the chow is supplemented with ezetimibe to block dietary sterol entry at the intestinal level (F shows area quantitations).

Fig. 8.

Glucose tolerance and adipose tissue fatty acid release in Abcg8 knockout mice. (A) Results of an intraperitoneal glucose tolerance in wild-type (open circles) or knockout mice (n = 4 per group); no differences were observed in the glucose excursions. Plasma baseline free fatty acid levels were significantly increased in the Abcg8 knockout mice (B, middle bar), and these were lowered back to wild-type levels in Abcg8 knockout mice whose diet was supplemented with ezetimibe (B, last bar). To further characterize the increase in fatty acids, adipocytes were isolated and cultured, and the release of fatty acids into media was examined under basal (−) and isoproterenol-stimulated (+) conditions (C). Basal levels of fatty acid release was indistinguishable between wild-type (open circles) and knockout adipocytes (C, filled circles, bottom two lines, marked by “−”). However, upon isoproterenol stimulation, significantly more fatty acid was released by adipocytes from abcg8 knockout mice (C, top two lines marked by “+” n = 5). A classical hormone-sensitive assay was performed on adipocyte homogenates (D), which showed that knockout adipocytes (n = 6) had higher lipolytic activity compared with wild-type adipocytes (n = 5).

Fig. 9.

Gene expression profiles of selected mRNAs in adipocytes isolated from Abcg8 knockout mice. Real-time PCR analyses for selected genes were performed from RNA isolated from adipose tissue from wild-type (open bars) or knockout mice (closed bars). Significantly increased gene expression patterns for Lxr, Abca1, Ldl-R (A), and Hsl (D) were noted in fat from knockout mice. Interestingly, sterol and fatty acid syntheses pathways did not seem to be perturbed (B,C).