Acetylation mediates taurocholate uptake in hepatocytes possibly through modulation of NTCP1 activity

In silico analysis of NTCP1 amino acid sequence

In silico analyzes were performed for the mouse NTCP1 protein sequence (362 aa), with two software. We first used Prediction of acetylation on internal lysines (PAIL) (http://bdmpail.biocuckoo.org/) as described in previous studies.¹³ This software facilitates the identification of possible acetylation sites in internal lysines with an accuracy of 85–89%. The internal lysines with a score greater than one predicted by the PAIL program in the NTCP1 amino acid sequence were visualized in the protein structure using the open-source tool Protter (http://wlab.ethz.ch/protter/start/).¹⁴

Mouse primary hepatocyte isolation

Mouse primary hepatocytes were isolated from male C57BL/6 mice of 8–12 weeks of age obtained from the Departamento de Investigación experimental y Bioterio at Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubiran (INCMNSZ). The procedures were performed in accordance with the Mexican Federal Regulation for Animal Experimentation and Care (NOM-062-ZOO-2001) and approved by the Animal Ethics Research Committee of INCMNSZ (Approval number 1560). Briefly, primary hepatocytes were obtained by in situ perfusion of the liver according to the method by Berry and Friend.¹⁵ Firstly, we cannulated and exsanguinated the liver in vivo followed by a continuous perfusion with collagenase. Secondly, we removed the liver and disaggregated the tissue in Hanks balanced salt solution (HBSS). Thirdly, we filtered the cell suspension through a 70 μM mesh and washed the filtered cells twice with HBSS. Finally, we re-suspended the cells in DMEM/F-12 supplemented with 1% antibiotic and 10% fetal bovine serum.

Nicotinamide incubation and taurocholate uptake assay

Firstly, we seeded hepatocytes into a 6-well or a 24-well CellBIND plate at 75% confluence and incubated at 37 °C. Unattached cells were removed by medium change after 4 hours. Cells were then treated with Nicotinamide 10 mM for 4 hours. After incubation, we used for western blot analysis cells in the 6-well plate as described below. On the other hand, cells in the 24-well plate were used to perform the taurocholate uptake assay.¹⁶ Briefly, hepatocytes were incubated in uptake buffer (D-glucose 11 mM, KCl 5.3 mM, CaCl₂ 1.8 mM, KH₂PO₄ 1.1 mM, HEPES 10 mM MgSO₄ 0.8 mM, pH 7.4) with or without sodium 136 mM in presence of sodium taurocholate 10 μM supplemented with 0.1 μCi of [G-³H]-taurocholic acid (specific activity 1–5 Ci/mmol, Perkin Elmer) for 20 min at 37 °C. We then washed the cells three times with PBS 1x, and lysed with SDS 0.05%. An aliquot of cell lysate was placed in a vial with scintillation liquid. We used a beta liquid scintillation counter to quantify the amount of ³H in the cell lysate.

Western blot analysis

Primary hepatocytes incubated with nicotinamide were homogenized in lysis buffer (Tris-HCl 50 mM, EDTA 1 mM, NaCl 150 mM, NP-40 1%) supplemented with phosphatase inhibitors (NaP₂O₇ 5 mM, Na₃VO₄ 1 mM, NaF 50 mM), protease inhibitors (Complete mini, Roche), and deacetylase inhibitors (Nicotinamide 5 mM, Sodium butyrate 1 mM). Homogenates were centrifuged 10,000 g, 10 minutes at 4 °C, and protein concentration in supernatants was measured using the Lowry method (BioRad). Western blot was performed using 40 μg of total protein. Proteins were separated with a 10% SDS-PAGE and transferred to a PVDF membrane. To evaluate the acetylated status of hepatic proteins, PVDF membranes were incubated with an antibody against acetylated lysines (#9441, Cell signaling). Proteins were detected using ultrasensitive horseradish peroxidase chemiluminescence (Pierce). A tubulin antibody (sc-7396 Santa Cruz Biotechnologies) was used for normalization.

Statistical analysis

The data are depicted as the mean ± standard error of the mean (SEM). Densitometry analysis of acetylated proteins was quantified by ImageJ PC software. To assure reproducibility, taurocholate uptake experiments were performed four times. Our results were analyzed using a one-way ANOVA or an unpaired t-test using GraphPad Prism program v 7.0a (GraphPad Prism, GraphPad Software, Inc. La Jolla CA). Differences were considered significant at P<0.05.

Prediction of acetylation sites on mouse NTCP1 amino acid sequence

To determine which internal lysines (K) on NTCP1 could be an acetylation target, the fasta sequence obtained from NCBI Slc10a1 [mus muculus] GenBank: AAH94023.1 was entered into the PAIL software. The in silico analysis showed eleven possible lysines that could be target of acetylation with a precision of 89.21%, four of these lysines had a score greater than one, which were K in positions 81, 113, 309 and 336. To locate these acetylation sites in the amino acid sequence and expected structure of NTCP1, we entered the NTCP1 amino acid sequence obtained from UniProt (UniProtKB-O08705) in Protter. The identified lysines were localized in the intracellular domains of NTCP1, except for K81 that was localized in an extracellular domain (Figure 1A).

Figure 1. Acetylation mediates taurocholate uptake in hepatocytes possibly through modulation of NTCP1 activity.

A) Predicted topological structure of mouse sodium and bile acid co-transporter NTCP1 generated using Protter v 1.0. We observe the seven transmembrane domains, and the extracellular amino-terminus and the intracellular carboxy-terminus identified by Phobius. High-throughput phosphorylation, ubiquitylation, and N-glycosylation sites are indicated according to phosphosite.com. The identified internal lysines by the in silico PAIL analysis as potential acetylation sites are highlighted in red. B) Global lysine acetylation in primary hepatocytes incubated with nicotinamide (NAM) 10 mM for 4 h. Lysates were analyzed by western blot using an anti-acetylated–lysine (AcK) antibody. The loading control was tubulin. Data are presented as mean ± SEM and * indicates a significant difference versus vehicle (V) at P < 0.05 by Student’s t-test. C) [³H]-Taurocholate uptake into primary hepatocytes was determined to evaluate NTCP1 activity. We performed four independent biological experiments with primary hepatocytes obtained from different C57BL6 mice and each experiment included at least 4 replicates. Significant difference is designated by letters were a > b at P < 0.05 by One-way ANOVA.

Effect of nicotinamide on protein acetylation and taurocholate uptake in mouse primary hepatocytes

The effect of acetylation on taurocholate uptake was evaluated in mouse primary hepatocytes treated with or without nicotinamide 10 mM for 4 hours. Nicotinamide incubation significantly increased acetylated proteins with respect to the control (Figure 1B). Unfortunately, we were unable to immunoprecipitate NTCP1 and to confirm whether NTCP1 was specifically acetylated. Nevertheless, when we evaluated taurocholate uptake, which depends mainly on NTCP1 activity, it was significantly increased in hepatocytes treated with NaCl 136 mM when compared to those without NaCl. Notably, when hepatocytes were previously treated with nicotinamide, we observed a significant reduction on taurocholate uptake (Figure 1C), suggesting that NTCP1 activity may depend on acetylation.

Underlying data

Figshare: Acetylation mediates taurocholate uptake in hepatocytes possibly through modulation of NTCP1 activity. https://doi.org/10.6084/m9.figshare.19226010.v2

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).