Active enterohepatic cycling is not required for the choleretic actions of 24-norUrsodeoxycholic acid in mice

Abstract

The pronounced choleretic properties of 24-norUrsodeoxycholic acid (norUDCA) to induce bicarbonate-rich bile secretion have been attributed to its ability to undergo cholehepatic shunting. The goal of this study was to identify the mechanisms underlying the choleretic actions of norUDCA and the role of the bile acid transporters. Here, we show that the apical sodium-dependent bile acid transporter (ASBT), organic solute transporter-α (OSTα), and organic anion transporting polypeptide 1a/1b (OATP1a/1b) transporters are dispensable for the norUDCA stimulation of bile flow and biliary bicarbonate secretion. Chloride channels in biliary epithelial cells provide the driving force for biliary secretion. In mouse large cholangiocytes, norUDCA potently stimulated chloride currents that were blocked by siRNA silencing and pharmacological inhibition of calcium-activated chloride channel transmembrane member 16A (TMEM16A) but unaffected by ASBT inhibition. In agreement, blocking intestinal bile acid reabsorption by coadministration of an ASBT inhibitor or bile acid sequestrant did not impact norUDCA stimulation of bile flow in WT mice. The results indicate that these major bile acid transporters are not directly involved in the absorption, cholehepatic shunting, or choleretic actions of norUDCA. Additionally, the findings support further investigation of the therapeutic synergy between norUDCA and ASBT inhibitors or bile acid sequestrants for cholestatic liver disease.

Keywords: Drug therapy; Gastroenterology; Hepatology; Toxins/drugs/xenobiotics; Transport.

Figure 1. norUDCA treatment increases bile flow and biliary bicarbonate and solute output in WT and Asbt^–/– mice.

(A) Bile flow. (B) Biliary bicarbonate concentration. (C) Bicarbonate output. (D) Biliary bile acid output. (E) Biliary bile acid species output (mean ± SEM). (F) Biliary bile acid composition expressed as pie charts. Unless indicated, median values (line), interquartile range (boxes), and minimum to maximum values (whiskers) are shown; n = 6–7 mice per group. For stacked bar graph, mean ± SD is shown. The data were evaluated for statistically significant differences using an ordinary 2-way ANOVA with a Tukey’s multiple-comparison test. Distinct lowercase letters indicate significant differences between groups (P < 0.05).

Figure 2. norUDCA treatment increases bile flow and biliary bicarbonate and solute output in WT and Osta^–/– Asbt^–/– mice.

(A) Bile flow. (B) Biliary bicarbonate concentration. (C) Bicarbonate output. (D) Glutathione concentration. (E) Glutathione output. Median values (line), interquartile range (boxes), and minimum to maximum values (whiskers) are shown; n = 5–7 mice per group. The data were evaluated for statistically significant differences using an ordinary 2-way ANOVA with a Tukey’s multiple-comparison test. Distinct lowercase letters indicate significant differences between groups (P < 0.05).

Figure 3. norUDCA treatment alters expression of a limited number of hepatic transporter genes.

RNA-Seq analysis of livers from WT mice fed chow or the norUDCA-diet. (A) Differentially expressed SLC membrane transporter genes whose expression was significantly induced (P < 0.05; n = 6 per group) in norUDCA-treated versus chow mice. (B) Differentially expressed ABC transporter and ATP P-type ATPase genes (P < 0.05; n = 6 per group) in the norUDCA-treated versus chow mice. (C) Hepatic expression of the indicated transporters and bile acid–related biosynthesis or metabolizing enzymes in WT and Asbt^–/– mice fed chow or the norUDCA-containing diet for 7 days. RNA was isolated from livers of individual mice and used for real-time PCR analysis. The mRNA expression was normalized using cyclophilin, and the results for each gene are expressed relative to chow-fed WT mice (set at 100%). Mean ± SD, n = 6–7 mice per group. The data were evaluated for statistically significant differences using an ordinary 1-way ANOVA with a Tukey’s multiple-comparison test. Distinct lowercase letters indicate significant differences between groups (P < 0.05).

Figure 4. norUDCA treatment increases bile flow and biliary bicarbonate and solute output in WT and Oatp1a/1b^–/– mice.

(A) Bile flow. (B) Biliary bicarbonate concentration. (C) Bicarbonate output. (D) Biliary pH. Median values (line), interquartile range (boxes), and minimum to maximum values (whiskers) are shown; n = 5 mice per group. The data were evaluated for statistically significant differences using an ordinary 2-way ANOVA with a Tukey’s multiple-comparison test. Distinct lowercase letters indicate significant differences between groups (P < 0.05).

Figure 5. norUDCA stimulates Cl^– currents mediated by TMEM16A.

(A) Representative whole-cell currents in MLC cells transfected with nontargeting siRNA or TMEM16A siRNA measured under basal conditions or during exposure to norUDCA (250 μM). Currents measured at –100 mV (black) or +100 mV (red), representing I_Cl^– are shown. Compound exposure is indicated by the black bar. A voltage-step protocol from a holding potential of –40 mV, with 450 ms steps from –100 to +100 mV in 20 mV increments. Currents demonstrated time-dependent activation at membrane potentials +100 mV. The I-V plot was generated from these protocols during basal (black) and norUDCA-stimulated (red) conditions. (B) Cumulative data demonstrating maximum increase in current density (pA/pF) in response to norUDCA in the absence or presence of TMEM16A siRNA silencing; n = 5–6 cells per group. Median values (line), interquartile range (boxes), and minimum to maximum values (whiskers) are shown. The data were evaluated for statistically significant differences using an ordinary 2-way ANOVA with a Tukey’s multiple-comparison test.

Figure 6. TMEM16A Cl^– current activation by norUDCA is independent of ASBT transport.

(A) Representative whole-cell currents in MLC cells measured under basal conditions and during exposure to norUDCA (250 μM) following preincubation with vehicle (top), TMEM16A inhibitor (10 μM A01; middle), or ASBTi (100 nM SC-435; bottom). Currents measured at –100 mV (black) or +100 mV (red) representing I_Cl^– are shown. Compound exposure is indicated by the black bar. The I-V plot was generated from these protocols during basal (black) and norUDCA-stimulated (red) conditions. (B) Cumulative data demonstrating maximum increase in current density (pA/pF) in response to norUDCA in the absence or presence of TMEM16A inhibitor or ASBTi; n = 5–35 cells per group. The data were evaluated for statistically significant differences using an ordinary 1-way ANOVA with a Tukey’s multiple-comparison test. Values with distinct superscript lowercase letters are significantly different (P < 0.05).

Figure 7. Pharmacological inhibition of intestinal bile acid absorption does not alter norUDCA induction of a bicarbonate-rich hypercholeresis in mice.

(A) Bile flow. (B) Biliary bicarbonate concentration. (C) Bicarbonate output. (D) Biliary pH. (E) Biliary bile acid concentration. (F) Biliary bile acid output. Median values (line), interquartile range (boxes), and minimum to maximum values (whiskers) are shown; n = 5 mice per group. The data were evaluated for statistically significant differences using an ordinary 2-way ANOVA with a Tukey’s multiple-comparison test. Distinct lowercase letters indicate significant differences between groups (P < 0.05).