Role of ABC transporters in secretion of cholesterol from liver into bile.

B y 1968, the basic principles underlying cholesterol solubility in bile had been established (1, 2). Free cholesterol (C) had been found to be almost entirely insoluble in water and very poorly soluble in bile salt solutions. Cholesterol was however very soluble in phosphotidylcholine (PC) lamellar liquid crystals (bilayers), and bile salts could solubilize the PC C liquid crystal bilayers into mixed micelles, thus carrying the C into solution. In 1970, I speculated (3) that the molecular mechanism for biliary lipid secretion was ‘‘that bile salts penetrate the cannilicular membrane from the interior of the liver cell and dissect out specifically lecithin and cholesterol leaving the membrane protein and other structural lipids intact.’’ This hypothesis, based on the physical chemistry of the lipid systems, has now been proven to be almost entirely incorrect. It is now clear that all biliary lipids are secreted in a controlled way by ABC transporters, and that the physical chemistry of the lipid interactions probably occurs within the lumen of the canniliculus and biliary ducts. The first break came with the knock out of the gene encoding the multiple drug-resistant protein MDR-2 (ABCB4), which prevented the secretion of PC and C into bile and caused liver pathology (4). High rates of bile salt infusion could not restore biliary PC in bile (5, 6), and it was concluded that MDR-2 was the PC transporter. Second, the bile salt export pump (BSEP) is the canniliculus-located ABC B11 gene product and is responsible for most of the bile salt transport from the hepatocyte into the bile cannilicular lumen (7–10). Defects in this gene gave rise to progressive familiar intrahepatic cholestatis type 2 (PFCI-2; ref. 11), whereas defects in MDR-2 (MDR-3 in humans) result in PFCI-3 (12). The final step in revealing the undeniable role of ABC proteins in the secretion of bile lipids appeared in a recent issue of PNAS (13). Helen Hobbs and coworkers disrupted ABCG5 and AC5G8 genes in mice and greatly inhibited C secretion into bile, showing that this ABC heterodimer is responsible for secretion of biliary C. Dr. Hobbs’ group had originally discovered ABCG5 G8 in their studies on a rare and mysterious disease, sitosterolemia, a recessive disorder characterized by poor biliary excretion of dietary sterols, increased absorption of plant and dietary sterols, hypercholesterolemia, hypersitosterolemia, and early coronary artherosclerosis. They found mutations in either of the two ABC monomers (ABCG5, ABCG8) in nine patients with sitosterolemia and concluded that these transporters ‘‘cooperate to limit intestinal absorption and promote biliary excretion of sterols’’ (14). Hobbs and coworkers then showed that ABCG5 and ABCG8 are present on nearly contiguous genes, and that when expressed, the proteins form a heterodimer in the endoplasmic reticulum. Coexpression is required for their movement into the Golgi and onto the apical surface of the cell. ABCB5 8 are expressed in the liver and intestine but not in other tissues (15). Next, they showed that, in transgenic animals overexpressing ABCB5 8, bile C is markedly increased, and that the absorption of added diet cholesterol and plant sterols is markedly decreased (16). In the study by Yu et al. (13), the genes for ABCG5 8 were disrupted, producing a G5G8 / mouse. On a chow diet (0.02% C, 0.05% sitosterol), the plasma sitosterol was elevated 30-fold, but C was decreased by 50%. When challenged with a 2% C diet, the plasma C increased 2.4fold in G5G8 / but not in controls. Liver C increased 3-fold in controls but 18-fold in G5G8 / mice. G5G8 / animals had decreased neutral fecal sterols and increased absorption of plant sterols, in a fashion similar to patients with sitosterolemia. Bile C was 5 nm ml in controls, but was low (0.1–0.64 nm ml) in G5G8 / mice. Heterozygotes had about half the biliary C. Biliary phospholipids and bile salts were virtually normal in G5G8 / animals. Yu et al. conclude ‘‘that the secretion of cholesterol does not couple quantitatively to the secretion of phospholipids.’’ On the high C diet, both control and G5G8 / animals increased biliary cholesterol from 5 to 14 nm ml in controls, and from 0.3 to 1.9 nm ml in G5G8 / mice. The increase in the G5G8 / mice indicates that high liver C can modestly increase non-ABCG5G8mediated C transport into bile. How does ABCG5 G8 transport C into bile? In G5G8 / animals, C in bile is decreased markedly (by 91% on average), even though the C acceptors, phospholipids, and bile salts are secreted normally. Transgenics overexpressing ABCG5G8 at moderate and high levels of transcript both greatly increase the C in bile without appreciably changing the bile salt and phospholipids (16). Wittenberg and Carey suggested that the lack of difference in excessive C secretion by transgenic mice expressing low and high amounts of G5G8 is caused by a saturation of the acceptors, PC and bile salt, in the bile canniliculus (17). The upper limit of C which could be secreted was between 1 and 1.4 mol of C per mol of phospholipids (16), which is quite similar to the maximum solubility of C in PC bilayers (1). Thus, ABCG5G8 moves C into acceptors until they are saturated. In many previous studies, bile salt was infused at different rates, and the bile salt, C, and PC secreted in bile was measured. C and PC were thought to be coupled, because increasing rates of infusion of bile acids caused secretion of more PC and C. The study on MDR-2 (ABCB4)-null animals also suggested that cholesterol was coupled with phospholipid secretion; not only were phospholipids absent, but C was very low in bile of MDR-2 / animals, whereas bile salt secretion was normal (4–6). This result could be related to the fact that PC is the major carrier of C, and when it is absent, C cannot move from the canniliculus into the aqueous phase even if murine bile salts are present. Infusion of hydrophilic bile salt tauroursodeoxycholete (TUDC) to reach secretion rates 1,200 nm min per 100 g was able to release 1.5 nm min per 100 g of C without any PC secretion (6). The ratio of 1:800 C:TUDC is well within the solubilizing capacity of TUDC alone. Perfusion of hydrophobic, human-like bile salts taurocholate and taurodeoxycholate to maximum levels produced a very small release of both PC and C into bile, 2 nm min per 100 g (5, 6). This finding indicates that bile salts are not effective in moving membrane C and PC into bile. Many years ago, we showed (1) that bile salts are extremely poor solubilizers of cholesterol, and that PC was required for micellar solubilization of C in bile. However, if the ratio of bile salts to C is 100, then some C can be solubilized without PC.