FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2195349961> ?p ?o ?g. }

• W2195349961 endingPage "1700" @default.
• W2195349961 startingPage "1691" @default.
• W2195349961 abstract "To distinguish the lithogenic effect of the classical estrogen receptor α (ERα) from that of the G protein-coupled receptor 30 (GPR30), a new estrogen receptor, on estrogen-induced gallstones, we investigated the entire spectrum of cholesterol crystallization pathways and sequences during the early stage of gallstone formation in gallbladder bile of ovariectomized female wild-type, GPR30(−/−), ERα(−/−), and GPR30(−/−)/ERα(−/−) mice treated with 17β-estradiol (E2) at 6 µg/day and fed a lithogenic diet for 12 days. E2 disrupted biliary cholesterol and bile salt metabolism through ERα and GPR30, leading to supersaturated bile and predisposing to the precipitation of cholesterol monohydrate crystals. In GPR30(−/−) mice, arc-like and tubular crystals formed first, followed by classical parallelogram-shaped cholesterol monohydrate crystals. In ERα(−/−) mice, precipitation of lamellar liquid crystals, typified by birefringent multilamellar vesicles, appeared earlier than cholesterol monohydrate crystals. Both crystallization pathways were accelerated in wild-type mice with the activation of GPR30 and ERα by E2. However, cholesterol crystallization was drastically retarded in GPR30(−/−)/ERα(−/−) mice. We concluded that E2 activates GPR30 and ERα to produce liquid crystalline versus anhydrous crystalline metastable intermediates evolving to cholesterol monohydrate crystals from supersaturated bile. GPR30 produces a synergistic lithogenic action with ERα to enhance E2-induced gallstone formation. To distinguish the lithogenic effect of the classical estrogen receptor α (ERα) from that of the G protein-coupled receptor 30 (GPR30), a new estrogen receptor, on estrogen-induced gallstones, we investigated the entire spectrum of cholesterol crystallization pathways and sequences during the early stage of gallstone formation in gallbladder bile of ovariectomized female wild-type, GPR30(−/−), ERα(−/−), and GPR30(−/−)/ERα(−/−) mice treated with 17β-estradiol (E2) at 6 µg/day and fed a lithogenic diet for 12 days. E2 disrupted biliary cholesterol and bile salt metabolism through ERα and GPR30, leading to supersaturated bile and predisposing to the precipitation of cholesterol monohydrate crystals. In GPR30(−/−) mice, arc-like and tubular crystals formed first, followed by classical parallelogram-shaped cholesterol monohydrate crystals. In ERα(−/−) mice, precipitation of lamellar liquid crystals, typified by birefringent multilamellar vesicles, appeared earlier than cholesterol monohydrate crystals. Both crystallization pathways were accelerated in wild-type mice with the activation of GPR30 and ERα by E2. However, cholesterol crystallization was drastically retarded in GPR30(−/−)/ERα(−/−) mice. We concluded that E2 activates GPR30 and ERα to produce liquid crystalline versus anhydrous crystalline metastable intermediates evolving to cholesterol monohydrate crystals from supersaturated bile. GPR30 produces a synergistic lithogenic action with ERα to enhance E2-induced gallstone formation. The precipitation of classical parallelogram-shaped cholesterol monohydrate crystals from supersaturated gallbladder bile is the first irreversible physical-chemical event during the early stage of cholesterol gallstone formation (1Wang D.Q. Cohen D.E. Carey M.C. Biliary lipids and cholesterol gallstone disease.J. Lipid Res. 2009; 50: S406-S411Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). It is well known that estrogen has a critical role in the pathogenesis of cholesterol cholelithiasis because gallstone prevalence is markedly higher in women than in men at all ages in every population studied (2Everhart J.E. Khare M. Hill M. Maurer K.R. Prevalence and ethnic differences in gallbladder disease in the United States.Gastroenterology. 1999; 117: 632-639Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). Many clinical studies have found that the use of oral contraceptives and conjugated estrogens in premenopausal and postmenopausal women significantly increases the prevalence of gallstones (3Everson G.T. McKinley C. Kern Jr, F. Mechanisms of gallstone formation in women. Effects of exogenous estrogen (Premarin) and dietary cholesterol on hepatic lipid metabolism.J. Clin. Invest. 1991; 87: 237-246Crossref PubMed Scopus (186) Google Scholar, 4Cirillo D.J. Wallace R.B. Rodabough R.J. Greenland P. LaCroix A.Z. Limacher M.C. Larson J.C. Effect of estrogen therapy on gallbladder disease.JAMA. 2005; 293: 330-339Crossref PubMed Scopus (200) Google Scholar, 5Thijs C. Knipschild P. Oral contraceptives and the risk of gallbladder disease: a meta-analysis.Am. J. Public Health. 1993; 83: 1113-1120Crossref PubMed Scopus (50) Google Scholar). Because elevated estrogen levels have a linear and positive relationship with the duration of gestation, the risk of gallstone formation becomes higher in the third trimester of pregnancy (6de Bari O. Wang T.Y. Liu M. Paik C.N. Portincasa P. Wang D.Q. Cholesterol cholelithiasis in pregnant women: pathogenesis, prevention and treatment.Ann. Hepatol. 2014; 13: 728-745Crossref PubMed Google Scholar). Increasing parity is a risk factor for gallstones, especially in younger women. Similar lithogenic effects are also found in men with prostatic cancer during estrogen therapy (7Henriksson P. Einarsson K. Eriksson A. Kelter U. Angelin B. Estrogen-induced gallstone formation in males. Relation to changes in serum and biliary lipids during hormonal treatment of prostatic carcinoma.J. Clin. Invest. 1989; 84: 811-816Crossref PubMed Scopus (86) Google Scholar, 8Angelin B. Olivecrona H. Reihner E. Rudling M. Stahlberg D. Eriksson M. Ewerth S. Henriksson P. Einarsson K. Hepatic cholesterol metabolism in estrogen-treated men.Gastroenterology. 1992; 103:: 1657-1663Abstract Full Text PDF PubMed Scopus (82) Google Scholar). These epidemiological and clinical studies have clearly demonstrated that high susceptibility to gallstone formation in women compared with men is related to differences in how the liver metabolizes cholesterol in response to estrogen (9de Bari O. Wang H.H. Portincasa P. Paik C.N. Liu M. Wang D.Q. Ezetimibe prevents the formation of oestrogen-induced cholesterol gallstones in mice.Eur. J. Clin. Invest. 2014; 44:: 1159-1168Crossref PubMed Scopus (10) Google Scholar). We have found that the classical estrogen receptor α (ERα), but not ERβ, in the liver plays a critical role in the pathogenesis of 17β-estradiol (E2)-induced gallstones in female mice (10Wang H.H. Afdhal N.H. Wang D.Q. Estrogen receptor α, but not β, plays a major role in 17β-estradiol-induced murine cholesterol gallstones.Gastroenterology. 2004; 127: 239-249Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Despite these observations, the metabolic abnormalities underlying the lithogenic effect of E2 on gallstone formation is not fully understood because the targeted deletion of the Erα gene cannot completely protect against gallstone formation in ovariectomized (OVX) female mice treated with high doses of E2 (11Wang H.H. Liu M. Clegg D.J. Portincasa P. Wang D.Q. New insights into the molecular mechanisms underlying effects of estrogen on cholesterol gallstone formation.Biochim. Biophys. Acta. 2009; 1791: 1037-1047Crossref PubMed Scopus (82) Google Scholar). Moreover, the discovery of the G protein-coupled receptor 30 (GPR30), a novel estrogen receptor, has led to a new question of whether it is involved in the lithogenic effect of E2 on gallstone formation (12Carmeci C. Thompson D.A. Ring H.Z. Francke U. Weigel R.J. Identification of a gene (GPR30) with homology to the G-protein-coupled receptor superfamily associated with estrogen receptor expression in breast cancer.Genomics. 1997; 45: 607-617Crossref PubMed Scopus (442) Google Scholar). Because E2 can efficiently bind to and activate both GPR30 and ERα, it is crucial to explore how E2, through GPR30, ERα, or both, influences the biliary and gallstone phenotypes. The genetic analysis with quantitative trait locus mapping techniques in mice supports the candidacy of Gpr30 for a new gallstone gene, Lith18 (13Lyons M.A. Korstanje R. Li R. Sheehan S.M. Walsh K.A. Rollins J.A. Carey M.C. Paigen B. Churchill G.A. Single and interacting QTLs for cholesterol gallstones revealed in an intercross between mouse strains NZB and SM.Mamm. Genome. 2005; 16: 152-163Crossref PubMed Scopus (27) Google Scholar, 14Lyons M.A. Wittenburg H. Cholesterol gallstone susceptibility loci: a mouse map, candidate gene evaluation, and guide to human LITH genes.Gastroenterology. 2006; 131: 1943-1970Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 15Wang H.H. Portincasa P. Afdhal N.H. Wang D.Q. Lith genes and genetic analysis of cholesterol gallstone formation.Gastroenterol. Clin. North Am. 2010; 39 (vii–viii.): 185-207Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). However, little information is available about the underlying lithogenic effect of GPR30 on gallstone formation. Thus, identifying the lithogenic mechanisms of GPR30 is now a focal point of interest. In the current study, to dissect the lithogenic effects of GPR30 and ERα on the formation of gallstones, we defined the entire spectrum of cholesterol crystallization pathways during the early stage of gallstone formation by investigating the formation of solid plate-like cholesterol monohydrate crystals and subsequent crystal growth in gallbladder bile of OVX female wild-type mice with intact expression of the Gpr30 and Erα genes as well as GPR30(−/−), ERα(−/−), and GPR30(−/−)/ERα(−/−) mice treated with E2 at 6 µg/day and fed a lithogenic diet for 12 days. We hypothesized that after GPR30 and ERα are activated by E2, GPR30 and ERα disrupt biliary cholesterol and bile salt metabolism through different mechanisms, leading to a failure of cholesterol solubilization in bile. As a result, these alterations induce a distinctly abnormal metastable physical-chemical state of gallbladder bile, thereby predisposing to the formation of solid cholesterol monohydrate crystals through different crystallization pathways. These studies would help distinguish the lithogenic effect of GPR30 from that of ERα, at a physical-chemical level, on enhancing cholelithogenesis. The inbred AKR/J mice of both genders purchased from the Jackson Laboratory (Bar Harbor, ME) were bred to generate female mice for the studies. Although AKR/J mice have been found to be a gallstone-resistant strain, they are still susceptible to E2-induced cholesterol gallstone formation (10Wang H.H. Afdhal N.H. Wang D.Q. Estrogen receptor α, but not β, plays a major role in 17β-estradiol-induced murine cholesterol gallstones.Gastroenterology. 2004; 127: 239-249Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Although AKR/J mice have intact expression of the Gpr30, Erα, and Erβ genes, expression levels of Erβ in the liver are almost undetectable under normal physiological conditions. Hepatic expression of Erβ is 50-fold lower than that of Erα even under the stimulation of E2. Thus, the AKR/J strain was used as control mice (i.e., wild-type mice). Other experimental animals included female GPR30(−/−), ERα(−/−), and GPR30(−/−)/ERα(−/−) mice, and all of these mice were on an AKR/J genetic background. We have established breeding colonies of these mice in-house. GPR30(−/−) mice were healthy and fertile. Of special note is that the ERα(+/−) heterozygotes were also fertile and showed no obvious phenotypes in association with the disrupted Erα genotype. A cross between heterozygous ERα(+/−) mice resulted in the live birth of normal litter sizes of homozygous ERα(−/−) mice. To generate GPR30(−/−)/ERα(−/−) mice, we have made a cross between GPR30(−/−) mice and heterozygous ERα(+/−) mice to create GPR30(−/−)/ERα(+/−) mice. After that, GPR30(−/−)/ERα(+/−) mice were bred to generate female GPR30(−/−)/ERα(−/−) mice for the studies. To exclude possible interindividual differences in endogenous estrogen concentrations, all experimental animals were gonadectomized and subsequently implanted with 17β-estradiol (E2)-releasing pellets (Innovative Research of America, Sarasota, FL). In brief, at 4 weeks of age, female mice were ovariectomized (OVX). At 8 weeks of age, the mice were implanted subcutaneously with pellets releasing E2 at 0 or 6 µg/day for 12 days. All animals were maintained in a temperature-controlled room (22 ± 1°C) with a 12-h day cycle (lights on from 0600 h to 1800 h) and were provided free access to water and normal mouse chow containing trace cholesterol (<0.02%) (Lab Rodent Diet, St. Louis, MO). To dissect the lithogenic effects of GPR30 and ERα on cholesterol crystallization during the early stage of gallstone formation, mice at 8 weeks old were fed the lithogenic diet containing 1% cholesterol, 15% butter fat, and 0.5% cholic acid for 12 days. All procedures were in accordance with current National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee of Saint Louis University (St. Louis, MO). After anesthetization with pentobarbital, a cholecystectomy was performed in overnight fasted mice (n = 5 per group for each time point) before (day 0, on chow) and at 3, 6, 9 and 12 days on the lithogenic diet. After a small hole was made in the fundus of the gallbladder, bulk bile dribbled by gravity, and mucin gels were pressed out digitally with the assistance of a 24 gauge needle. The entire gallbladder bile sample was placed on a glass slide at room temperature (∼22°C) and observed without a cover slip using a polarizing light microscope and then with a cover slip using phase contrast optics. These gallbladder bile samples were examined by microscopic analysis for the presence of mucin gels, liquid crystals, solid cholesterol monohydrate crystals, sandy stones, and real gallstones according to previously published criteria (16Wang D.Q. Paigen B. Carey M.C. Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: physical-chemistry of gallbladder bile.J. Lipid Res. 1997; 38: 1395-1411Abstract Full Text PDF PubMed Google Scholar). Mucin gels were observed as nonbirefringent amorphous strands. Arc-like and tubular crystals (assumed to be metastable transitional forms of anhydrous cholesterol being hydrated to cholesterol monohydrate crystals), plate-like cholesterol monohydrate crystals, as well as small, aggregated, and fused liquid crystals were defined according to previously established criteria (17Wang D.Q. Carey M.C. Complete mapping of crystallization pathways during cholesterol precipitation from model bile: influence of physical-chemical variables of pathophysiologic relevance and identification of a stable liquid crystalline state in cold, dilute and hydrophilic bile salt-containing systems.J. Lipid Res. 1996; 37: 606-630Abstract Full Text PDF PubMed Google Scholar). The images of cholesterol monohydrate crystals were analyzed by a Carl Zeiss Imaging System with an AxioVision Rel 4.6 software (Carl Zeiss Microimaging, GmbH Göttingen, Germany). After microscopic analysis, gallbladder bile was collected, frozen, and stored at –20°C for lipid studies. Biliary cholesterol was determined using an enzymatic assay (18Fromm H. Amin P. Klein H. Kupke I. Use of a simple enzymatic assay for cholesterol analysis in human bile.J. Lipid Res. 1980; 21: 259-261Abstract Full Text PDF PubMed Google Scholar). Biliary phospholipids were measured as inorganic phosphorus by the method of Bartlett (19Bartlett G.R. Phosphorus assay in column chromatography.J. Biol. Chem. 1959; 234: 466-468Abstract Full Text PDF PubMed Google Scholar). Total bile salt concentration was measured enzymatically by the 3α-hydroxysteroid dehydrogenase method (20Turley S.D. Dietschy J.M. Re-evaluation of the 3 alpha-hydroxysteroid dehydrogenase assay for total bile acids in bile.J. Lipid Res. 1978; 19: 924-928Abstract Full Text PDF PubMed Google Scholar). Individual bile salt species were determined by HPLC (21Rossi S.S. Converse J.L. Hofmann A.F. High pressure liquid chromatographic analysis of conjugated bile acids in human bile: simultaneous resolution of sulfated and unsulfated lithocholyl amidates and the common conjugated bile acids.J. Lipid Res. 1987; 28: 589-595Abstract Full Text PDF PubMed Google Scholar). Cholesterol saturation index (CSI) of pooled gallbladder bile was calculated from critical tables (22Carey M.C. Critical tables for calculating the cholesterol saturation of native bile.J. Lipid Res. 1978; 19: 945-955Abstract Full Text PDF PubMed Google Scholar) that were established for taurocholate, the predominant bile salt in bile of mice on the lithogenic diet. The hydrophobicity index of bile samples was calculated according to Heuman's method (23Heuman D.M. Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions.J. Lipid Res. 1989; 30: 719-730Abstract Full Text PDF PubMed Google Scholar). Relative lipid compositions of pooled gallbladder bile (n = 5 per group at each time point) were plotted on condensed phased diagrams. For graphic analysis, the phase limits of the micellar zones and the crystallization pathways were extrapolated from model systems developed for taurocholate at 37°C and at a total lipid concentration of 9 g/l (17Wang D.Q. Carey M.C. Complete mapping of crystallization pathways during cholesterol precipitation from model bile: influence of physical-chemical variables of pathophysiologic relevance and identification of a stable liquid crystalline state in cold, dilute and hydrophilic bile salt-containing systems.J. Lipid Res. 1996; 37: 606-630Abstract Full Text PDF PubMed Google Scholar). At 12 days after E2 treatment and the lithogenic diet feeding, a dynamic measurement of gallbladder emptying function was performed in mice (n = 4 per group) in response to a high-fat meal according to previously published methods (24Wang H.H. Portincasa P. Liu M. Tso P. Samuelson L.C. Wang D.Q. Effect of gallbladder hypomotility on cholesterol crystallization and growth in CCK-deficient mice.Biochim. Biophys. Acta. 2010; 1801: 138-146Crossref PubMed Scopus (37) Google Scholar). After mice were fasted overnight but had free access to water, they were anesthetized with pentobarbital. During laparotomy, a PE-10 polyethylene catheter was inserted into the duodenum. The duodenal catheter was externalized through the left abdominal wall and connected to an infusion pump (Kent Scientific, Litchfield, CT). After all the surgical procedures were completed, the gallbladder was clearly exposed, and its volume was carefully measured with a micro-caliper. By assuming an ellipsoid shape of the organ, fasting gallbladder volume was calculated using the following formula: Gallbladder volume (μl) = length (mm) × width (mm) × depth (mm) × π/6. To measure postprandial gallbladder volume, mice were intraduodenally infused with corn oil (i.e., a fatty meal) at 40 µl/min for 5 min. At 30 min after the corn oil infusion, gallbladder volume was carefully measured again with a micro-caliper. Gallbladder emptying function was calculated by a difference in gallbladder volume before and after the duodenal infusion of corn oil. Representative blocks of paraffin-embedded gallbladder tissues were cut at 4 μm thickness, dewaxed, and rehydrated. For GPR30 and ERα staining, antigens were retrieved by boiling in 10 mM citrate buffer (pH 7.0) for 1 min. All the staining processes were performed by using a Histostain Plus 3rd Gen IHC Detection Kit according to the manufacturer's instructions (Invitrogen, Camarillo, CA). The sections were incubated with anti-GPR30 or anti-ERα antibodies (Santa Cruz, Dallas, TX) at a dilution of 1:100. The primary antibodies were replaced by the blocking solution containing 10% nonimmune goat serum for negative control slides. After washing, the sections were incubated with the corresponding secondary antibodies for 30 min at room temperature. Subsequently, the sections were counterstained with hematoxylin, dehydrated through an alcohol series to xylene, and mounted. Total RNA was extracted from gallbladder tissues of mice (n = 4 per group) using RNeasy Mini (Qiagen, Valencia, CA). Reverse transcription reaction was performed using the iScript Reverse Transcription Supermix for quantitative RT-PCR (Bio-Rad, Hercules, CA) with 1 μg of total RNA and random hexamers to generate cDNA. Primer Express Software (Applied Biosystems, Foster City, CA) was used to design the primers based on sequence data available from GenBank. Supplementary Table 1 lists the sequences of the primers for the genes below. The mRNA levels of mucin gene 1 (Muc1), Muc2, Muc3, Muc4, Muc5ac, Muc5b, cholecystokinin-1 receptor (Cck-1r), acyl-CoA:cholesterol acyltransferase, isoform 2 (Acat2), scavenger receptor class B, member 1 (Sr-b1), ATP-binding cassette transporter g5 (Abcg5), Abcg8, and Abca1 in the gallbladder were examined in triplicate by quantitative real-time PCR assays (25Wang H.H. Afdhal N.H. Wang D.Q. Overexpression of estrogen receptor alpha increases hepatic cholesterogenesis, leading to biliary hypersecretion in mice.J. Lipid Res. 2006; 47: 778-786Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Relative mRNA levels were calculated using the threshold cycle of an unknown sample against a standard curve with known copy numbers. To obtain a normalized target value, the target amount was divided by the endogenous reference amount of mouse β-Actin as internal control. All data are expressed as mean ± SD. Statistically significant differences among groups of mice were assessed by Student's t-test, Mann-Whitney U-tests, or Chi-square tests. If the F-value was significant, comparisons among groups of mice were further analyzed by a multiple comparison test. Analyses were performed with SuperANOVA software (Abacus Concepts, Berkeley, CA). Statistical significance was defined as a two-tailed probability of less than 0.05. As shown in Fig. 1, during the 12-day period of E2 treatment and the lithogenic diet feeding, multiple types of cholesterol crystals formed in OVX GPR30(−/−) mice, with arc-like and tubular crystals of anhydrous cholesterol evolving to the classical notched rhombohedral cholesterol monohydrate crystals. By contrast, solid plate-like cholesterol monohydrate crystals precipitated from the liquid crystalline pathway in OVX ERα(−/−) mice. Both the anhydrous crystalline and the liquid crystalline habits were observed in OVX wild-type mice. However, no liquid crystals or solid cholesterol crystals were found in OVX GPR30(−/−)/ERα(−/−) mice. These results indicated that the activation of GPR30 by E2 promoted the precipitation of solid cholesterol monohydrate crystals from liquid crystals independently of ERα because the latter induced cholesterol crystallization from the anhydrous crystalline pathway. Figure 2 illustrates cholesterol crystallization pathways and sequences in gallbladder bile of OVX mice treated with E2 as functions of days on the lithogenic diet. In OVX wild-type mice, a thick layer of sticky mucin gels adherent to the gallbladder wall was first observed, followed by liquid crystals and anhydrous cholesterol crystals. At day 9, cholesterol monohydrate crystals were present with mucin gels. At day 12, solid cholesterol monohydrate crystals were consolidated by mucin gels as agglomerates. In OVX GPR30(−/−) mice, mucin gels appeared first, followed by anhydrous cholesterol crystals and then cholesterol monohydrate crystals. As expected, mucin gels appeared first in OVX ERα(−/−) mice. Subsequently, aggregated and fused liquid crystals were found at day 9, and many cholesterol monohydrate crystals precipitated in bile at day 12. We found that after 12 days on the lithogenic diet, the number of solid cholesterol monohydrate crystals was markedly greater in OVX wild-type mice than in OVX GPR30(−/−) and ERα(−/−) mice. Notably, only a thin layer of mucin gels appeared at day 9 in OVX GPR30(−/−)/ERα(−/−) mice. No liquid crystals or solid cholesterol crystals were detected in these mice at 12 days. These results indicated that the time of cholesterol crystallization was greatly prolonged in OVX GPR30(−/−)/ERα(−/−) mice. Table 1 lists the relative biliary lipid compositions of pooled gallbladder bile in OVX mice treated with E2 as well as before (day 0, on chow) and during feeding the lithogenic diet for 12 days. For phase analysis, a group of truncated phase diagrams was created for pooled gallbladder bile of OVX mice during the 12-day period of E2 treatment and the lithogenic diet feeding (Fig. 3). In the lithogenic state, relative lipid compositions of pooled gallbladder bile gradually moved upward and to the right of the phase diagrams in all groups of OVX mice. Such a shift was caused by a relative increase in cholesterol and phospholipid concentrations and a relative decrease in bile salt concentrations. In OVX wild-type mice, relative biliary lipid compositions of bile at day 0 and day 3 were located within the micellar zone, whereas after day 6, relative biliary lipid compositions passed through region B and entered region C. By phase analysis, the bile was composed of two or three phases, namely saturated micelles, solid cholesterol crystals, or plus liquid crystals. In OVX GPR30(−/−) mice, relative biliary lipid compositions of bile at day 9 entered region B, in which arc-like and tubular crystals appeared before cholesterol monohydrate crystals. During lithogenesis, relative lipid compositions of bile in OVX ERα(−/−) mice directly entered region C from the micellar zone. As expected, liquid crystals invariably preceded cholesterol monohydrate crystals during crystallization. Of note, relative biliary lipid composition of bile in OVX GPR30(−/−)/ERα(−/−) mice still stayed in the micellar zone. By phase analysis, the bile consisted of only unsaturated micelles, indicating that gallbladder bile was unsaturated.TABLE 1Biliary lipid compositions of pooled gallbladder bile during cholesterol crystallizationDayCh (mol%)PL (mol%)BS (mol%)Ch/PL RatioCh/BS Ratio[TL] (g/dl)CSIWild-type02.4711.5086.030.2150.0298.940.5633.2812.6884.030.2590.0398.770.7065.0313.3181.660.3780.0629.451.0195.7415.1579.120.3790.0739.461.05126.8316.2776.910.4200.0899.841.17GPR30(−/−)01.9810.6887.340.1850.0238.760.4732.2913.0584.660.1750.0278.690.4863.5614.2782.170.2500.0438.670.7095.1613.9680.880.3700.0648.811.01126.1015.7778.130.3870.07810.481.06ERα(−/−)01.8710.6887.450.1750.0218.820.4432.5413.0084.470.1950.0308.940.5363.5413.1483.320.2690.0428.730.7395.5216.0878.390.3430.0708.041.00125.9416.2077.860.3670.0769.861.02GPR30(−/−)/ERα(−/−)01.5511.3587.090.1370.0188.220.3632.1912.0685.750.1810.0258.470.4962.7815.0582.170.1840.0348.350.5393.3915.9180.700.2130.0428.490.62124.0917.4578.460.2340.0529.260.69Values were determined from pooled gallbladder bile (n = 5 per group). BS, bile salts; Ch, cholesterol; CSI, cholesterol saturation index; PL, phospholipids; [TL], total lipid concentration. Open table in a new tab Values were determined from pooled gallbladder bile (n = 5 per group). BS, bile salts; Ch, cholesterol; CSI, cholesterol saturation index; PL, phospholipids; [TL], total lipid concentration. Analysis of individual bile salt species by HPLC showed that all bile salts in gallbladder bile of E2-treated mice were taurine conjugated with a similar distribution of bile salt composition. As expected, in these mice fed the lithogenic diet, taurocholate (50.5–55.5%) was the major bile salt of biliary pool, followed by taurochenodeoxycholate (13.3–32.4%). There was a low concentration in tauro-β-muricholate (4.5–13.9%), taurodeoxycholate (6.0–9.6%), tauro-ω-muricholate (0.9–7.2%), and tauroursodeoxycholate (3.2–5.5%). Hydrophobicity indexes of bile salts in bile were comparable (–0.08 to +0.14) among four groups of mice. These results indicate that the deletion of either the Erα or the Gpr30 gene alone, or both did not influence bile salt species in bile. At 12 days on the lithogenic diet, E2 treatment caused the largest fasting gallbladder volumes in OVX wild-type mice, followed by OVX GPR30(−/−), ERα(−/−), and GPR30(−/−)/ERα(−/−) mice (Fig. 4). We also measured postprandial gallbladder volumes in these mice in response to a fatty meal. We observed that a significant portion of gallbladder bile was emptied out in OVX GPR30(−/−)/ERα(−/−) mice in response to the fatty meal. However, it was not the case in OVX wild-type, GPR30(−/−), and ERα(−/−) mice, suggesting an impaired gallbladder emptying function in these mice. Bile stasis induced by hypomobile gallbladder provides the time necessary to accommodate growth and agglomeration of solid cholesterol crystals into microlithiasis entrapped within mucin gels. Therefore, gallbladder hypomotility is an important risk factor for gallstone formation (26Portincasa P. Di Ciaula A. Wang H.H. Palasciano G. van Erpecum K.J. Moschetta A. Wang D.Q. Coordinate regulation of gallbladder motor function in the gut-liver axis.Hepatology. 2008; 47: 2112-2126Crossref PubMed Scopus (104) Google Scholar). Figure 5 displays three modes of growth habits of solid cholesterol crystals, as observed in E2-treated OVX wild-type mice with the highest CSI value. Figure 5A shows the most common crystal growth habit: proportional enlargement patterns in which solid cholesterol crystals grow bidirectionally (i.e., by both length and width). The second mode of crystal growth habit was spiral dislocation growth, with the pyramidal surface containing numerous spiral layers crystallizing by a screw dislocation (Fig. 5B). The third mode of crystal growth habit was twin crystal growth, with the crystals growing upright and perpendicular to the surface (Fig. 5C). All of these crystal growth habits led to a rapid enlargement of solid cholesterol crystals in size. As reveale" @default.
• W2195349961 created "2016-06-24" @default.
• W2195349961 creator A5015149955 @default.
• W2195349961 creator A5017353282 @default.
• W2195349961 creator A5019115247 @default.
• W2195349961 creator A5052244609 @default.
• W2195349961 creator A5088702595 @default.
• W2195349961 date "2015-09-01" @default.
• W2195349961 modified "2023-10-18" @default.
• W2195349961 title "Estrogen induces two distinct cholesterol crystallization pathways by activating ERα and GPR30 in female mice" @default.
• W2195349961 cites W1513897103 @default.
• W2195349961 cites W1533289312 @default.
• W2195349961 cites W1555140191 @default.
• W2195349961 cites W1830716241 @default.
• W2195349961 cites W1858850028 @default.
• W2195349961 cites W1895325632 @default.
• W2195349961 cites W1915358754 @default.
• W2195349961 cites W1929425341 @default.
• W2195349961 cites W1943976550 @default.
• W2195349961 cites W1968671012 @default.
• W2195349961 cites W1970498015 @default.
• W2195349961 cites W1972250939 @default.
• W2195349961 cites W1981467972 @default.
• W2195349961 cites W2009924670 @default.
• W2195349961 cites W2016019734 @default.
• W2195349961 cites W2040355234 @default.
• W2195349961 cites W2042338884 @default.
• W2195349961 cites W2049116118 @default.
• W2195349961 cites W2052764939 @default.
• W2195349961 cites W2052774670 @default.
• W2195349961 cites W2062791955 @default.
• W2195349961 cites W2063474196 @default.
• W2195349961 cites W2070443889 @default.
• W2195349961 cites W2076261621 @default.
• W2195349961 cites W2077347937 @default.
• W2195349961 cites W2084659900 @default.
• W2195349961 cites W2104661100 @default.
• W2195349961 cites W2107479916 @default.
• W2195349961 cites W2107913761 @default.
• W2195349961 cites W2117749613 @default.
• W2195349961 cites W2123036265 @default.
• W2195349961 cites W2123973543 @default.
• W2195349961 cites W2127924075 @default.
• W2195349961 cites W2128559420 @default.
• W2195349961 cites W2132932170 @default.
• W2195349961 cites W2139213251 @default.
• W2195349961 cites W2141697699 @default.
• W2195349961 cites W2152438501 @default.
• W2195349961 doi "https://doi.org/10.1194/jlr.m059121" @default.
• W2195349961 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4548773" @default.
• W2195349961 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/26152119" @default.
• W2195349961 hasPublicationYear "2015" @default.
• W2195349961 type Work @default.
• W2195349961 sameAs 2195349961 @default.
• W2195349961 citedByCount "35" @default.
• W2195349961 countsByYear W21953499612016 @default.
• W2195349961 countsByYear W21953499612017 @default.
• W2195349961 countsByYear W21953499612018 @default.
• W2195349961 countsByYear W21953499612019 @default.
• W2195349961 countsByYear W21953499612020 @default.
• W2195349961 countsByYear W21953499612021 @default.
• W2195349961 countsByYear W21953499612022 @default.
• W2195349961 countsByYear W21953499612023 @default.
• W2195349961 crossrefType "journal-article" @default.
• W2195349961 hasAuthorship W2195349961A5015149955 @default.
• W2195349961 hasAuthorship W2195349961A5017353282 @default.
• W2195349961 hasAuthorship W2195349961A5019115247 @default.
• W2195349961 hasAuthorship W2195349961A5052244609 @default.
• W2195349961 hasAuthorship W2195349961A5088702595 @default.
• W2195349961 hasBestOaLocation W21953499611 @default.
• W2195349961 hasConcept C121608353 @default.
• W2195349961 hasConcept C126322002 @default.
• W2195349961 hasConcept C134018914 @default.
• W2195349961 hasConcept C166471694 @default.
• W2195349961 hasConcept C185592680 @default.
• W2195349961 hasConcept C2777164284 @default.
• W2195349961 hasConcept C2778163477 @default.
• W2195349961 hasConcept C530470458 @default.
• W2195349961 hasConcept C71924100 @default.
• W2195349961 hasConcept C84606932 @default.
• W2195349961 hasConcept C86803240 @default.
• W2195349961 hasConcept C95444343 @default.
• W2195349961 hasConceptScore W2195349961C121608353 @default.
• W2195349961 hasConceptScore W2195349961C126322002 @default.
• W2195349961 hasConceptScore W2195349961C134018914 @default.
• W2195349961 hasConceptScore W2195349961C166471694 @default.
• W2195349961 hasConceptScore W2195349961C185592680 @default.
• W2195349961 hasConceptScore W2195349961C2777164284 @default.
• W2195349961 hasConceptScore W2195349961C2778163477 @default.
• W2195349961 hasConceptScore W2195349961C530470458 @default.
• W2195349961 hasConceptScore W2195349961C71924100 @default.
• W2195349961 hasConceptScore W2195349961C84606932 @default.
• W2195349961 hasConceptScore W2195349961C86803240 @default.
• W2195349961 hasConceptScore W2195349961C95444343 @default.
• W2195349961 hasIssue "9" @default.
• W2195349961 hasLocation W21953499611 @default.
• W2195349961 hasLocation W21953499612 @default.
• W2195349961 hasLocation W21953499613 @default.

• next