A. K. Groen

Regulation of biliary lipid secretion by mdr2 P-glycoprotein in the mouse.

Disruption of the mdr2 gene in mice leads to a complete absence of phospholipid from bile (Smit, J. J. M., et al. 1993. Cell. 75:451-462). We have investigated the control of both mdr2 P-glycoprotein (Pgp) expression and bile salt secretion on biliary lipid secretion in the mouse. Lipid secretion was monitored at various bile salt output rates in wild-type mice (+/+), heterozygotes (+/-), and homozygotes (-/-) for mdr2 gene disruption. In (-/-) mice, phospholipid secretion was negligible at all bile salt output rates. In (+/-) mice, a curvilinear relation between bile salt and phospholipid secretion was observed similar to that in (+/+) mice; however, at all bile salt secretion rates phospholipid secretion was reduced compared to (+/+) mice, indicating that mdr2 Pgp exerts a strong control over secretion. Infusion of increasing amounts of taurocholate up to maximal secretory rate led to a decline in the phospholipid and cholesterol secretion in both (+/+) and (+/-) mice in accordance to what has been observed in other species. In contrast, in (-/-) mice cholesterol secretion increased under these conditions while phospholipid output remained extremely low. The increased cholesterol secretion may represent extraction of cholesterol from the canalicular plasma membrane by taurocholate micelles as opposed to the concomitant secretion of both phospholipid and cholesterol in the presence of a functional mdr2 Pgp. Increased bile flow in (-/-) mice could be attributed completely to an increase in the bile salt-independent fraction and may therefore be caused by the bile duct proliferation in these mice.

Complementary functions of the flippase ATP8B1 and the floppase ABCB4 in maintaining canalicular membrane integrity.

BACKGROUND & AIMS Progressive familial intrahepatic cholestasis can be caused by mutations in ABCB4 or ATP8B1; each encodes a protein that translocates phospholipids, but in opposite directions. ABCB4 flops phosphatidylcholine from the inner to the outer leaflet, where it is extracted by bile salts. ATP8B1, in complex with the accessory protein CDC50A, flips phosphatidylserine in the reverse direction. Abcb4(-/-) mice lack biliary secretion of phosphatidylcholine, whereas Atp8b1-deficient mice have increased excretion of phosphatidylserine into bile. Each system is thought to have a role protecting the canalicular membrane from bile salts. METHODS To investigate the relationship between the mechanisms of ABCB4 and ATP8B1, we expressed the transporters separately and together in cultured cells and studied viability and phospholipid transport. We also created mice with disruptions in ABCB4 and ATP8B1 (double knockouts) and studied bile formation and hepatic damage in mice fed bile salts. RESULTS Overexpression of ABCB4 was toxic to HEK293T cells; the toxicity was counteracted by coexpression of the ATP8B1-CDC50A complex. In Atp8b1-deficient mice, bile salts induced extraction of phosphatidylserine and ectoenzymes from the canalicular membrane; this process was not observed in the double-knockout mice. CONCLUSIONS ATP8B1 is required for hepatocyte function, particularly in the presence of ABCB4. This is most likely because the phosphatidylserine flippase complex of ATP8B1-CDC50A counteracts the destabilization of the membrane that occurs when ABCB4 flops phosphatidylcholine. Lipid asymmetry is therefore important for the integrity of the canalicular membrane; ABCB4 and ATP8B1 cooperate to protect hepatocytes from bile salts.

Class III P-glycoproteins mediate the formation of lipoprotein X in the mouse.

Cholestasis is associated with hypercholesterolemia and appearance of the abnormal lipoprotein X (LpX) in plasma. Using mice with a disrupted Mdr2 gene, we tested the hypothesis that LpX originates as a biliary lipid vesicle. Mdr2-deficient mice lack Mdr2 P-glycoprotein, the canalicular translocator for phosphatidylcholine, and secrete virtually no phospholipid and cholesterol in bile. Bile duct ligation of Mdr2(+)/+ mice induced a dramatic increase in the plasma cholesterol and phospholipid concentration. Agarose electrophoresis, density gradient ultracentrifugation, gel permeation, and electron microscopy revealed that the majority of phospholipid and cholesterol was present as LpX, a 40-100 nm vesicle with an aqueous lumen. In contrast, the plasma cholesterol and phospholipid concentration in Mdr2(-)/- mice decreased upon bile duct ligation, and plasma fractionation revealed a complete absence of LpX. In mice with various expression levels of Mdr2 or MDR3, the human homolog of Mdr2, we observed that the plasma level of cholesterol and phospholipid during cholestasis correlated very closely with the expression level of these canalicular P-glycoproteins. These data demonstrate that during cholestasis there is a quantitative shift of lipid secretion from bile to the plasma compartment in the form of LpX. The concentration of this lipoprotein is determined by the activity of the canalicular phospholipid translocator.

Apolipoprotein M predicts pre-beta-HDL formation: studies in type 2 diabetic and nondiabetic subjects

Objective.  Studies in mice suggest that plasma apoM is lowered in hyperinsulinaemic diabetes and that apoM stimulates formation of pre‐β‐HDL. Pre‐β‐HDL is an acceptor of cellular cholesterol and may be critical for reverse cholesterol transport. Herein, we examined whether patients with type 2 diabetes have reduced plasma apoM and whether apoM is associated with pre‐β‐HDL formation and cellular cholesterol efflux.

Abcg5/8 Independent Biliary Cholesterol Excretion in Atp8b1-Deficient Mice

BACKGROUNDS & AIMS ATP8B1 is a phosphatidylserine flippase in the canalicular membrane; patients with mutations in ATP8B1 develop severe chronic (PFIC1) or periodic (BRIC1) cholestatic liver disease. We have observed that Atp8b1 deficiency leads to enhanced biliary cholesterol excretion. It has been established that biliary cholesterol excretion depends on transport by the heterodimer Abcg5/Abcg8. We hypothesized that the increased cholesterol output was due to enhanced extraction from the altered canalicular membrane rather than to higher Abcg5/Abcg8 activity. We therefore studied the relation between Abcg5/Abcg8 expression and biliary cholesterol excretion in mice lacking Atp8b1, Abcg8, or both (GF mice). METHODS Bile formation was studied in LXR agonist-fed wild-type mice as well as mice lacking Atp8b1 or Abcg8, or in GF mice upon infusion of taurocholate. Bile samples were analyzed for cholesterol, bile salt, phospholipids, and ectoenzyme content. RESULTS LXR agonist increased Abcg5/8 expression, and this was accompanied by increased biliary cholesterol output in both wild-type and Atp8b1(G308V/G308V) mice. However, Atp8b1(G308V/G308V) mice maintained higher cholesterol output. Although in Abcg8(-/-) mice biliary cholesterol output was severely reduced, GF mice displayed high biliary cholesterol output, which was comparable with wild-type mice. Bile of both Atp8b1(G308V/G308V) and GF mice displayed elevated levels of phosphatidylserine and sphingomyelin, indicating membrane stress. CONCLUSIONS Our data demonstrate that the increased biliary cholesterol excretion in Atp8b1-deficient mice is independent of Abcg5/8 activity. This implicates that Atp8b1 deficiency leads to a decrease in the detergent resistance and subsequent nonspecific extraction of cholesterol from the canalicular membrane by bile salts.

Distinct immuno-localization of mucin and other biliary proteins in human cholesterol gallstones

BACKGROUND/AIMS Cholesterol gallstones consist of cholesterol crystals and smaller amounts of pigments and calcium salts, arrayed on a mucin plus protein matrix. The localization of the various biliary proteins in the stones has not been characterized. We aimed to localize several biliary proteins in gallstones in order to determine their possible role in stone formation and growth. METHODS The distribution of several matrix proteins and their relationships to the minerals were determined using immunostaining and EDAX microanalysis on hemisected cholesterol gallstones. RESULTS Pigment areas were rich in calcium and contained Cu, P and S. These elements were absent in cholesterol regions. Mucin was identified in a three-dimensional network intercalated between cholesterol crystals and as septa between deposits of pigments and cholesterol; APF/CBP and ApN coated only the pigment deposits. No specific topographical localization was found for albumin or IgA. CONCLUSIONS This suggests a role for mucin, APF/ CBP and ApN in the formation of cholesterol gallstones. We propose that cholesterol crystals bind directly to mucin, whereas calcium salts and pigments deposit on APF/CBP and ApN bind to the mucin.

Regulation of MDR2 P-glycoprotein expression by bile salts

The phosphatidyl translocating activity of the mdr2 P-glycoprotein (Pgp) in the canalicular membrane of the mouse hepatocyte is a rate-controlling step in the biliary secretion of phospholipid. Since bile salts also regulate the secretion of biliary lipids, we investigated the influence of the type of bile salt in the circulation on mdr2 Pgp expression and activity. Male mice were led a purified diet to which either 0.1% (w/w) cholate or 0.5% (w/w) ursodeoxycholate was added. This led to a near-complete replacement of the endogenous bile salt pool (mainly tauromuricholate) by taurocholate or tauroursodeoxycholate respectively. The phospholipid secretion capacity was then determined by infusion of increasing amounts of tauroursodeoxycholate. Cholate feeding resulted in a 55% increase in maximal phospholipid secretion compared with that in mice on the control diet. Northern blotting revealed that cholate feeding increased mdr2 Pgp mRNA levels by 42%. Feeding with ursodeoxycholate did not influence the maximum rate of phospholipid output or the mdr2 mRNA content. Female mice had a higher basal mdr2 Pgp mRNA level than male mice, and this was also correlated with a higher phospholipid secretion capacity. This could be explained by the 4-fold higher basal cholate content in the bile of female compared with male mice. Our results suggest that the type of bile salts in the circulation influences the expression of the mdr2 gene.