Estrogen receptor α, but not β, plays a major role in 17β-estradiol-induced murine cholesterol gallstones  Helen H. Wang, Nezam H. Afdhal, David Q.-H. Wang  Gastroenterology  Volume 127, Issue 1, Pages 239-249 (July 2004) DOI: 10.1053/j.gastro.2004.03.059

Figure 1 Gallstone prevalence at 12 weeks of feeding the lithogenic diet in gonadally intact or gonadectomized AKR mice of both genders treated for 12 weeks (A) with E2 (a full agonist for both ERα and β) at 0, 3, or 6 μg/day, and (B) with ICI 182,780 (a novel, pure, and full estrogen antagonist for both ERα and β), PPT (a highly selective agonist for ERα), DPN (a full agonist for ERβ), and tamoxifen (a synthetic, nonsteroidal, trans-isomeric derivative of triphenylethylene displaying estrogen-like activity). ∗P < 0.01, and ∗∗P < 0.05 compared with mice with intact gonads. Gastroenterology 2004 127, 239-249DOI: (10.1053/j.gastro.2004.03.059)

Figure 2 Outputs (μmol/h/kg/body weight) of cholesterol, phospholipid, and bile salts during the first hour of biliary secretion at 12 weeks of feeding the lithogenic diet. (A) Outputs of 3 biliary lipids show a dose-dependent increase in gonadectomized AKR mice of both genders treated with exogenous E2. (B) Compared with mice with intact gonads, outputs of biliary cholesterol are augmented significantly (P < 0.001) in gonadectomized mice receiving PPT or tamoxifen. However, DPN does not influence outputs of biliary lipids. Most notably, the E2 (6 μg/day) effects on biliary cholesterol output were abolished by the antiestrogenic ICI 182,780 compared with the use of E2 alone. Gastroenterology 2004 127, 239-249DOI: (10.1053/j.gastro.2004.03.059)

Figure 3 Effects of estrogen and its antagonists and agonists on bile flow rates at 12 weeks of feeding the lithogenic diet. Bile flow rates (43–59 μL/min/kg) are similar among mice with intact gonads, the E2-deficient mice, and mice treated with E2 at 3 μg/day, or DPN. However, bile flow rates are increased to 95–105 μL/min/kg by E2 at 6 μg/day, to 88–90 μL/min/kg by PPT, and to 77–79 μL/min/kg by tamoxifen. Of note is that the choleretic effects of E2 are blocked fully by ICI 182,780, and bile flow rates are reduced to 50–52 μL/min/kg. ∗P < 0.001 compared with mice with intact gonads. Gastroenterology 2004 127, 239-249DOI: (10.1053/j.gastro.2004.03.059)

Figure 4 Bile flow as a function of bile salt output in female AKR mice at 12 weeks of feeding the lithogenic diet. Each point represents bile flow and bile salt output measurements in the same sample during 8-hour periods of biliary washout. No gender differences were found (male data not shown). (A) Bile salt-dependent bile flow in mice receiving E2 at 6 μg/day is significantly (P < 0.01) greater than that in mice receiving E2 at 3 μg/day, mice with intact ovaries, and the E2-deficient mice. Equations for canalicular bile salt-dependent flow are y =120 (±51) + 39 (±6) x (r = 0.91; P < 0.001) for mice receiving E2 at 6 μg/day; y = 124 (±60) + 25 (±6) x (r = 0.90; P < 0.001) for mice receiving E2 at 3 μg/day; y = 140 (±73) + 20 (±5) x (r = 0.88; P < 0.001) for mice with intact ovaries; and y = 100 (±42) + 19 (±4) x (r = 0.89; P < 0.001) for the E2-deficient mice. On extrapolation of the regression lines to the ordinate, the y intercepts are similar in these 4 groups of mice, suggesting that bile salt-independent flow is similar. (B) Bile salt-dependent bile flow in mice treated with PPT and tamoxifen (TAM) is significantly (P < 0.001) greater than that in mice treated with DPN and ICI 182,780. Equations for canalicular bile salt-dependent flow are y = 135 (±48) + 35 (±6) x (r = 0.90; P < 0.001) for the PPT group; y = 122 (±51) + 32 (±5) x (r = 0.90; P < 0.001) for the tamoxifen (TAM) group; y = 150 (±69) + 19 (±4) x (r = 0.89; P < 0.001) for the DPN group; and y = 120 (±52) + 18 (±3) x (r = 0.91; P < 0.001) for the ICI 182,780 group. Again, bile salt-independent flow is essentially similar in these 4 groups of mice. Gastroenterology 2004 127, 239-249DOI: (10.1053/j.gastro.2004.03.059)

Figure 5 Relative mRNA levels of the hepatic Erα and Erβ genes in mice fed the lithogenic diet for 12 weeks. Most notably, expression levels of (A) Erα are ∼50-fold higher than those of (B) Erβ. The relative mRNA levels for Erα are similar among mice with intact gonads, the E2-deficient mice, and mice receiving E2 at 3 μg/day. In contrast, expression levels of Erα are increased significantly (P < 0.01) in mice treated with E2 at 6 μg/day and tamoxifen. However, the E2 effects on Erα expression are blocked completely by ICI 182,780. The ERα-selective agonist PPT increases expression levels of Erα only. Expression levels of Erβ are slightly (P = NS) up-regulated by E2 at 6 μg/day and the ERβ-selective agonist DPN, but not by tamoxifen and E2 at 0 or 3 μg/day. Gastroenterology 2004 127, 239-249DOI: (10.1053/j.gastro.2004.03.059)

Figure 6 Protein concentrations of hepatic ERα and ERβ in gonadally intact or gonadectomized AKR mice of both genders fed the lithogenic diet for 12 weeks. Representative Western blots for ERα and ERβ are shown for female AKR mice. The data are expressed relative to the protein level of ERα and ERβ in female mice with intact ovaries, and their protein concentrations are set at 1. (A) Protein concentrations of ERα are similar among mice with intact gonads, the E2-deficient mice, and mice receiving E2 at 3 μg/day. In contrast, the long-term administration of E2 at 6 μg/day, ICI 182,780, PPT, DPN, and tamoxifen significantly (P < 0.01) decreases protein concentrations of ERα. (B) The long-term treatment of ER ligands also reduces protein concentrations of ERβ; however, these alterations do not reach significant statistical differences compared with mice with intact gonads and mice receiving E2 at 0 or 3 μg/day. Gastroenterology 2004 127, 239-249DOI: (10.1053/j.gastro.2004.03.059)