Role of hepatic transporters in prevention of bile acid toxicity after partial hepatectomy in mice

Abstract

The enterohepatic recirculation of bile acids (BAs) is important in several physiological processes. Although there has been considerable research on liver regeneration after two-thirds partial hepatectomy (PHx), little is known about how the liver protects itself against BA toxicity during regeneration. In this study, various BAs in plasma and liver, the composition of micelle-forming bile constituents, as well as gene expression of the main hepatobiliary transporters were quantified in sham-operated and PHx mice 24 and 48 h after surgery. PHx did not influence the hepatic concentrations of taurine-conjugated BAs (T-BA) but increased the concentration of glycine-conjugated (G-BA) and unconjugated BAs. Total BA excretion (microg x min(-1) x g liver wt(-1)) increased 2.4-fold and was accompanied by a 55% increase in bile flow after PHx. The plasma concentrations of T-BAs (402-fold), G-BAs (17-fold), and unconjugated BAs (500-fold) increased. The mRNA and protein levels of the BA uptake transporter Ntcp were unchanged after PHx, whereas the canalicular Bsep protein increased twofold at 48 h. The basolateral efflux transporter Mrp3 was induced at the mRNA (2.6-fold) and protein (3.1-fold) levels after PHx, which may contribute to elevated plasma BA and bilirubin levels. Biliary phospholipid excretion was nearly doubled in PHx mice, most likely owing to increased mRNA expression of the phospholipid transporter, Mdr2. In conclusion, the remnant liver after PHx excretes 2.5-fold more BAs and three times more phospholipids per gram liver than the sham-operated mouse liver. Upregulation of phospholipid transport may be important in protecting the biliary tract from BA toxicity during PHx.

Fig. 1.

Effect of partial hepatectomy (PHx) on plasma alkaline phosphatase (ALP), alanine aminotransferase (ALT), and conjugated bilirubin. In a preliminary ALP time course study, blood samples were collected at several time points between 15 min and 14 days after surgery, whereas to measure ALT and conjugated bilirubin the blood samples were collected at 24 and 48 h after surgery. Data are presented as means ± SE of 5–6 mice, except for the activity of ALP at 24 and 48 h, data represents means ± SE of 11–12 mice, because the preliminary and the final experiment values were combined at these 2 time points after it was determined that they were not statistically different. *Significant difference (P < 0.05) from the respective value of the sham-operated mice.

Fig. 2.

Effect of PHx on hepatic and serum concentrations of unconjugated (A and E), glycine-conjugated (G-; B and F), taurine-conjugated (T-; C and G), and total (D and H) bile acids in mice. Bars represent means ± SE of 5–6 mice. MCA, muricholic acids; CA, cholic acid; UDCA, ursodeoxycholic acid; CDCA, chenodeoxycholic acid; LCA, lithocholic acid. *Statistically significant difference (P < 0.05) from the respective value of the sham-operated mice. †Statistically significant changes (P < 0.05) in sham-operated mice 24 to 48 h.

Fig. 3.

Effect of PHx on bile flow per kilogram body weight (top) and bile flow per gram liver weight (bottom) in mice. PHx or sham operations were performed on mice, and bile was collected for 60 min at 24 or 48 h after surgery. Bars represent means ± SE of 5–6 mice. BW, body weight; LW, liver weight. *Significant difference (P < 0.05) from the respective value of the sham-operated mice.

Fig. 4.

Effect of PHx on biliary concentration, biliary excretion/body weight, and biliary excretion/liver weight of unconjugated (Unconj-), glycine-conjugated, and taurine-conjugated bile acids (BA). Biliary concentration (A, D, G, J), biliary excretion/liver weight (B, E, H, K), and biliary excretion/body weight (C, F, I, L) of unconjugated-, glycine-, and taurine-conjugated bile acids were quantified in mice following PHx or sham operations. Bile was collected for 60 min 24 or 48 h after surgery. The 24- and 48-h data were combined in sham and PHx groups after it was determined that they were not statistically different. Bars represent means ± SE of 10–12 mice. *Significant difference (P < 0.05) from the respective value of the sham-operated mice.

Fig. 5.

Effect of PHx on biliary concentration, biliary excretion/liver weight, and biliary excretion/body weight of glutathione (A–C), phospholipids (D–F), and cholesterol (G–I) in mice. Bars represent means ± SE of 5–6 mice. *Significant difference (P < 0.05) from the respective value of the sham-operated mice.

Fig. 6.

Effect of PHx on mRNA expression of canalicular transporters Mdr2, Abcg5, Abcg8, Mrp2, Bsep, and Atp8b1 in mouse liver. Bars represent means of relative light unit (RLU)/10 μg total RNA ± SE of 5–6 mice. *Significant difference (P < 0.05) from the respective value of the sham-operated mice.

Fig. 7.

Effect of PHx on mRNA expression of basolateral efflux transporter Mrp3, Mrp4, Ostα, and Ostβ in mouse liver. Bars represent means of relative light unit 10 μg total RNA ± SE of 5–8 mice. *Significant difference (P < 0.05) from the respective value of the sham-operated mice.

Fig. 8.

Bsep, Mrp2, Mrp3, and Ostα protein abundance in livers of mice 24 and 48 h following sham and PHx surgery. Western blots for Bsep (∼170 kDa), Mrp2 (∼190 kDa), Mrp3 (∼180 kDa), and Ostα (∼40 kDa) were performed with use of liver membrane protein fraction (40 μg protein/lane) from sham- and PHx-operated mice 24 and 48 h after surgery. β-Actin (∼45 kDa) blot was used as a protein loading control for each transporter. Bars represent the relative Bsep, Mrp2, Mrp3, or Ostα protein amounts ± SE of 5–8 mice and as representative blots at 48 h. The scales for mean relative protein expression differ for each transport protein. *Statistical difference (P < 0.05) from the respective values of sham-operated mice.

Fig. 9.

Immunofluorescent analysis of Bsep, Mrp2, and Mrp3 in mouse liver 48 h following sham and PHx surgeries. Indirect immunofluorescence against canalicular Bsep and Mrp2 and sinusoidal Mrp3 (green) was conducted on liver cryosections (5 μm) obtained 48 h following sham and PHx surgery. Representative periportal regions are shown. Portions of images were enlarged and provided as insets. Representative images are shown. Bar, 50 μm.

Fig. 10.

Effect of PHx on expression of basolateral uptake transporters Oatp1a1, Oatp1a4, Oatp1b2, and Ntcp mRNA in mouse liver. Bars represent means of unit (RLU) 10 μg total RNA ± SE of 5–8 mice. *Significant difference (P < 0.05) from the respective value of the sham-operated mice.

Fig. 11.

Oatp1a1, Oatp1a4, Oatp 1b2, and Ntcp protein abundance in livers of mice 24 and 48 h following sham and PHx surgery. Western blots for Oatp1a1 (∼70 kDa), Oatp1a4 (∼70 kDa), Oatp1b2 (∼75 kDa), and Ntcp (∼50 kDa) were performed with use of liver membrane protein fractions (40 μg protein/lane) from sham- and PHx-operated mice 24 and 48 h after surgery. β-Actin (∼45 kDa) blot was used as a protein loading control for each transporter. Bars represent the Oatp1a1, Oatp1a4, Oatp1b2, or Ntcp protein expression ± SE of 5–6 mice, and representative blots at 48 h are shown. In the case of Oatp1a4, the 24-h representative blot is shown. The scales for mean relative protein amount differ for each transport protein. *Statistical difference (P < 0.05) from the respective values of sham-operated mice.

Fig. 12.

Summary figure depicting the consequences of PHx on bile acid homeostasis and transporters in mice. Arrows represent changes from wild-type mice for a metabolite, enzyme, or transporter. PHx does not significantly influence the hepatic concentrations of taurine-conjugated BAs and, consequently, the concentrations of total BAs in the liver. However, the glycine and unconjugated BA concentrations increase significantly in liver. To maintain BA concentrations after PHx, the remnant liver, besides decreasing bile acid de novo synthesis (Cyp7a, 8b1, 27a1), increases the BA efflux into bile, probably via increasing Bsep activity. After PHx, hepatocytes also limit the accumulation of BAs by upregulating basolateral efflux transporters, including Mrp3 and Ostα/β. Probably owing to this aforementioned induction of transporters, the plasma concentrations of unconjugated, glycine-, and taurine-conjugated BAs increase. The biliary tree is likely protected against the harmful effects of enhanced bile acid excretion by Mdr2, which can be responsible for enhanced canalicular excretion of phospholipids. Conj, conjugated.