Activation of Estrogen Receptor G Protein-Coupled Receptor 30 Enhances Cholesterol Cholelithogenesis in Female Mice

Abstract

Background and aims: Estrogen is an important risk factor for cholesterol gallstone disease because women are twice as likely as men to form gallstones. The classical estrogen receptor α (ERα), but not ERβ, in the liver plays a critical role in the formation of estrogen-induced gallstones in female mice. The molecular mechanisms underlying the lithogenic effect of estrogen on gallstone formation have become more complicated with the identification of G protein-coupled receptor 30 (GPR30), an estrogen receptor.

Approach and results: We investigated the biliary and gallstone phenotypes in ovariectomized female GPR30^-/- , ERα^-/- , and wild-type mice injected intramuscularly with the potent GPR30-selective agonist G-1 at 0 or 1 μg/day and fed a lithogenic diet for 8 weeks. The activation of GPR30 by G-1 enhanced cholelithogenesis by suppressing expression of cholesterol 7α-hydroxylase, the rate-limiting enzyme for the classical pathway of bile salt synthesis. These metabolic abnormalities led to an increase in biliary cholesterol concentrations in company with hepatic hyposecretion of biliary bile salts, thereby inducing cholesterol-supersaturated gallbladder bile and accelerating cholesterol crystallization. G-1 also impairs gallbladder emptying, leading to sluggish gallbladder motility and promoting the development of biliary sludge in the early stage of gallstone formation. The prevalence rates of gallstones were 80% in wild-type and ERα^-/- mice treated with G-1 compared to 10% in wild-type mice receiving no G-1. However, no gallstones were formed in GPR30^-/- mice treated with G-1.

Conclusions: GPR30 produces additional lithogenic actions, working independently of ERα, to increase susceptible to gallstone formation in female mice; both GPR30 and ERα are potential therapeutic targets for cholesterol gallstone disease, particularly in women and patients exposed to high levels of estrogen.

FIG. 1.

(A) At 8 weeks on the lithogenic diet, the prevalent rates of gallstones are 10% in OVX wild-type mice receiving no G-1, and most of these mice form solid cholesterol monohydrate crystals. The GPR30-selective agonist G-1 promotes gallstone formation in 80% of OVX wild-type and ERα^−/− mice. Notably, only solid cholesterol monohydrate crystals, but not gallstones, are found in OVX GPR30^−/− mice even treated with G-1 for 8 weeks. (B) Representative photomicrographs of solid plate-like cholesterol monohydrate crystals and gallstones as observed by polarizing light microscopy in gallbladder bile of OVX wild-type, GPR30^−/−, and ERα^−/− mice injected intramuscularly with G-1 at 0 or 1 μg/day and fed the lithogenic diet for 8 weeks. Magnifications are ×800 for left two panels and ×200 for right two panels.

FIG. 2.

Effects of G-1 on the relative biliary lipid composition of pooled gallbladder bile at 8 weeks on the lithogenic diet, as plotted on a condensed phase diagram according to a total lipid concentration (~10 g/dL) of the bile. One-phase micellar zone at the bottom is enclosed by a solid curved line. Above it, two solid and two dashed lines divide the phase diagram into regions A-E with different crystallization pathways. After being fed the lithogenic diet for 8 weeks, the relative biliary lipid composition of pooled gallbladder bile in OVX wild-type mice receiving no G-1 and in OVX GPR30^−/− mice treated with G-1 at 1 μg/day is located on the borderline between the one-phase micellar zone and left two-phase region B. By phase analysis, gallbladder bile is supersaturated with cholesterol. By contrast, G-1 administration to OVX wild-type and ERα^−/− mice leads to the relative biliary lipid composition of pooled gallbladder bile plotted in the central three-phase zone denoted region C, where bile is composed of solid cholesterol monohydrate crystals, liquid crystals, and saturated micelles at equilibrium. The symbol ▲ represents the relative biliary lipid composition of pooled gallbladder bile at 8 weeks on the lithogenic diet in OVX wild-type mice receiving no G-1, as well as ■, ♦, and ● for OVX wild-type, GPR30^−/−, and ERα^−/− mice treated with G-1 at 1 μg/day, respectively.

FIG. 3.

Effects of G-1 on bile flow rates (A) as well as hepatic output of biliary cholesterol (B), phospholipids (C), and bile salts (D) during the first hour of biliary secretion in OVX wild-type, GPR30^−/−, and ERα^−/− mice treated with G-1 at 0 or 1 μg/day and fed the lithogenic diet for 8 weeks. ^#P < 0.05 compared to OVX wild-type mice receiving no G-1 and fed the lithogenic diet for 8 weeks; *P < 0.05 compared to OVX wild-type mice treated with G-1 at 1 μg/day and fed the lithogenic diet for 8 weeks. Abbreviation: B.W., body weight.

FIG. 4.

Gallbladder emptying rates in response to exogenous CCK-8 stimulation (as indicated by the arrows) during the early stage of gallstone formation in OVX mice treated with G-1 at 0 or 1 μg/day and fed the lithogenic diet for 2 weeks (left panels). PBS solution is used as control. Right panels show representative photomicrographs of mucin gels and biliary sludge as observed by polarizing light microscopy. Fresh gallbladder bile is examined in the same groups of OVX mice immediately after the gallbladder emptying study. G-1 impairs gallbladder contractile function in OVX wild-type and ERα^−/− mice, leading to the formation of biliary sludge containing numerous plate-like cholesterol monohydrate crystals. By contrast, only mucin gels and small liquid crystals, but not solid cholesterol crystals, are found in OVX wild-type mice receiving no G-1 and GPR30^−/− mice even treated with G-1 for 2 weeks. Magnifications are ×800 for all photos. *P < 0.01 and **P < 0.001 compared with PBS solution.

FIG. 5.

Effects of G-1 on mRNA levels of Gpr30, Erα, and Erβ in the liver. The relative mRNA levels of these genes in OVX wild-type mice receiving no G-1 and fed the lithogenic diet for 8 weeks are set at 1. Compared to OVX wild-type mice receiving no G-1, expression of Gpr30 in the liver is significantly increased in OVX wild-type and ERα (−/−), but not GPR30 (−/−), mice treated with G-1 at 1 μg/day and fed the lithogenic diet for 8 weeks. However, G-1 treatment does not influence expression of hepatic Erα or Erβ in these four groups of OVX mice. *P < 0.01 compared to OVX wild-type mice receiving no G-1 and fed the lithogenic diet for 8 weeks.

FIG. 6.

Effects of G-1 on mRNA levels of the genes involved in the regulation of hepatic lipid metabolism in the lithogenic state. The relative mRNA levels of the genes in OVX wild-type mice receiving no G-1 and fed the lithogenic diet for 8 weeks are set at 1. The data show the mRNA levels of the genes encoding hepatic lipid transporters, enzymes for the regulation of bile salt and cholesterol synthesis, and lipoprotein receptors in OVX wild-type, GPR30^−/−, and ERα^−/− mice treated with G-1 at 0 or 1 μg/day and fed the lithogenic diet for 8 weeks. *P < 0.01 compared with OVX wild-type mice receiving no G-1 and fed the lithogenic diet for 8 weeks. See text for further description and for abbreviations.

FIG. 7.

Working model for a possible G-1–GPR30–CYP7A1 pathway enhancing cholelithogenesis. We found that the GPR30-selective agonist G-1 activates hepatic Gpr30 expression, with the latter suppressing activity of Cyp7a1, the rate-limiting enzyme for the classical pathway of bile salt synthesis. As a result, these changes lead to increased biliary cholesterol concentration and hepatic bile salt hyposecretion, thereby reducing cholesterol solubility in bile and causing cholesterol-supersaturated bile that predisposes to solid cholesterol crystal precipitation and gallstone formation. Abbreviations: BS, bile salts; Ch, cholesterol; PL, phospholipids.