Evidence that the adenosine triphosphate-binding cassette G5/G8-independent pathway plays a determinant role in cholesterol gallstone formation in mice

Abstract

The adenosine triphosphate-binding cassette (ABC) sterol transporter, Abcg5/g8, is Lith9 in mice, and two gallstone-associated variants in ABCG5/G8 have been identified in humans. Although ABCG5/G8 plays a critical role in determining hepatic sterol secretion, cholesterol is still secreted to bile in sitosterolemic patients with a defect in either ABCG5 or ABCG8 and in either Abcg5/g8 double- or single-knockout mice. We hypothesize that in the defect of ABCG5/G8, an ABCG5/G8-independent pathway is essential for regulating hepatic secretion of biliary sterols, which is independent of the lithogenic mechanism of the ABCG5/G8 pathway. To elucidate the effect of the ABCG5/G8-independent pathway on cholelithogenesis, we investigated the biliary and gallstone characteristics in male wild-type (WT), ABCG5(-/-)/G8(-/-), and ABCG8 (-/-) mice fed a lithogenic diet or varying amounts of cholesterol, treated with a liver X receptor (LXR) agonist, or injected intravenously with [(3) H]sitostanol- and [(14) C]cholesterol-labeled high-density lipoprotein (HDL). We found that ABCG5(-/-)/G8(-/-) and ABCG8 (-/-) mice displayed the same biliary and gallstone phenotypes. Although both groups of knockout mice showed a significant reduction in hepatic cholesterol output compared to WT mice, they still formed gallstones. The LXR agonist significantly increased biliary cholesterol secretion and gallstones in WT, but not ABCG5(-/-)/G8(-/-) or ABCG8 (-/-), mice. The 6-hour recovery of [(14) C]cholesterol in hepatic bile was significantly lower in both groups of knockout mice than in WT mice and [(3) H]sitostanol was detected in WT, but not ABCG5(-/-)/G8(-/-) or ABCG8 (-/-), mice.

Conclusions: The ABCG5/G8-independent pathway plays an important role in regulating biliary cholesterol secretion, the transport of HDL-derived cholesterol from plasma to bile, and gallstone formation, which works independently of the ABCG5/G8 pathway. Further studies are needed to observe whether this pathway is also operational in humans. (Hepatology 2016;64:853-864).

Figure 1

(A) CSI values of pooled gallbladder bile as a function of days on the lithogenic diet in WT, ABCG5(−/−)/G8(−/−), and ABCG8 (−/−) mice. (B) Photomicrographs of cholesterol monohydrate crystal and gallstone formation observed in mice after 14, 28, and 56 days on the lithogenic diet. Notably, although ABCG5(−/−)/G8(−/−) and ABCG8 (−/−) mice displayed significantly slower crystallization, growth, and agglomeration of cholesterol monohydrate crystals compared to WT mice, they still formed gallstones. All magnifications are 800x by polarizing light microscopy, except for the panels (400x) of day 28 for WT mice and of day 56 for ABCG5(−/−)/G8(−/−) and ABCG8 (−/−) mice, and the panel (200x) of day 56 for WT mice. (C) Gallstone prevalence was significantly higher in WT mice than in ABCG5(−/−)/G8(−/−) and ABCG8 (−/−) mice and (D) the frequency of stone number was assessed in these mice after 56 days on the lithogenic diet.

Figure 2

Relative lipid composition (moles per 100 moles) of pooled gallbladder bile (n=5 per group) at each time point (0, 14, 28, and 56 days on the lithogenic diet) is plotted on condensed phase diagrams in which regions A to E with different crystallization sequences have been described elsewhere (27). With passage of time, relative biliary lipid composition of pooled gallbladder bile gradually moves upward and to the right and enters the crystallization region C from the micellar zone in three groups of mice. This indicates that gallbladder bile reaches supersaturation not only in WT mice, but also in ABCG5(−/−)/G8(−/−) and ABCG8 (−/−) mice after 14 days on the lithogenic diet.

Figure 3

(A) Hepatic output of biliary cholesterol and phospholipids, but not bile salts, as well as ratios of cholesterol/phospholipids and cholesterol/bile salts were significantly reduced in ABCG5(−/−)/G8(−/−) and ABCG8 (−/−) mice compared to those in WT mice fed the lithogenic diet for 56 days. Bile flow was comparable among three groups of mice. (B) There was a dose-dependent increase in mRNA levels and protein concentrations of ABCG5 and ABCG8 in WT mice in response to an increase in dietary cholesterol. (C) Hepatic cholesterol secretion was increased in a dose-dependent manner not only in WT mice, but also in ABCG5(−/−)/G8(−/−) and ABCG8 (−/−) mice although biliary cholesterol output was lower in the latter than in the former. Abbreviations: BS, bile salts; Ch, cholesterol; PL, phospholipids.

Figure 4

(A) Relative lipid composition of individual hepatic bile (n=5 each per group) is plotted on condensed phase diagrams for average total lipid concentration of 3 g/dL. This system exhibits the same physical states at equilibrium as for gallbladder bile, but all crystallization pathways are shifted to left with decreases in total lipid concentration and the one-phase micellar zone becomes smaller (27). These changes generate new condensed phase diagrams, with enlarged region E. Relative lipid composition of all hepatic bile is located in region E, where the bile is composed of liquid crystals and saturated micelles but not solid cholesterol crystals at equilibrium. After 56 days of the lithogenic diet feeding, (B) cholesterol/phospholipid ratios, CSI values, and total lipid concentrations of hepatic bile were significantly reduced in ABCG5(−/−)/G8(−/−) and ABCG8 (−/−) mice compared to those in WT mice. (C) Bile salt species of hepatic bile and (D) hydrophobic index of bile salt pool, as well as (E) expression of hepatic Cyp7a1 and Cyp27a1 were comparable among three groups of mice. See text for further details. Abbreviations: Ch, cholesterol; CSI, cholesterol saturation index; PL, phospholipids.

Figure 5

(A) By 6 hours after the injection of [¹⁴C]cholesterol-labeled HDL, cumulative radioactivity recovered in hepatic bile was significantly higher in WT mice than in ABCG5(−/−)/G8(−/−) and ABCG8 (−/−) mice. However, the HDL-derived [³H]sitostanol was found in WT, but not ABCG5(−/−)/G8(−/−) or ABCG8 (−/−) mice. The LXR agonist T0901317 significantly increased (B) mRNA levels and (C) protein concentrations of ABCG5 and ABCG8 in the liver of WT mice. The LXR agonist significantly (D) augmented hepatic cholesterol output and (E) promoted gallstone formation in WT, but not ABCG5(−/−)/G8(−/−) or ABCG8 (−/−) mice.

Figure 6

(A) Fasting gallbladder volumes as a function of days on the lithogenic diet. (B) Cholesterol and cholesteryl ester concentrations in the gallbladder tissues after 56 days of the lithogenic diet feeding. (C) Postprandial gallbladder emptying rates in response to duodenal infusion of corn oil or exogenously administered CCK-8 via intravenous injection (as indicated by the arrows) during the early stage of gallstone formation in mice fed the lithogenic diet for 14 days. Gallbladder emptying was impaired in WT, ABCG5(−/−)/G8(−/−), and ABCG8 (−/−) mice. (D) The mRNA levels of the genes involved in gallbladder lipid metabolism and transporters. See text for further description.

Figure 7

(A) In the basal state, ~65% and ~35% of biliary cholesterol output are determined by the ABCG5/G8 and the ABCG5/G8-independent pathways, respectively. The cholesterol molecules secreted by these two pathways form biliary vesicles with phospholipids secreted by the ABCB4 transporter. Bile salts are secreted by the ABCB11 transporter, forming simple and mixed micelles with varying amounts of cholesterol and phospholipids. (B) When ABCG5/G8 in the liver is disrupted, hepatic cholesterol output is significantly reduced and the ABCG5/G8-independent pathway plays a major role in regulating biliary cholesterol secretion. Notably, the resulting vesicles contain less cholesterol. (C) In the lithogenic state, excess amounts of cholesterol are secreted to bile by the liver through the ABCG5/G8-independent pathway under conditions of disruption of ABCG5/G8, leading to supersaturated bile. These abnormalities promote cholesterol crystallization and gallstone formation in ABCG5(−/−)/G8(−/−) and ABCG8 (−/−) mice. Abbreviations: ABC, ATP-binding cassette (transporter); BS, bile salts; Ch, cholesterol; LXR, liver X receptor; PL, phospholipids.