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Quantitative atlas of cytochrome P450, UDP-glucuronosyltransferase, and transporter proteins in jejunum of morbidly obese subjects Eisuke Miyauchi, Masanori Tachikawa, Xavier Decleves, Yasuo Uchida, JeanLuc Bouillot, Christine Poitou, Jean-Michel Oppert, Stéphane Mouly, Jean-François Bergmann, Tetsuya Terasaki, Jean-Michel Scherrmann, and Célia Lloret-Linares Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00085 • Publication Date (Web): 27 Jun 2016 Downloaded from http://pubs.acs.org on July 2, 2016

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Molecular Pharmaceutics

Quantitative atlas of cytochrome P450, UDP-glucuronosyltransferase, and transporter proteins in jejunum of morbidly obese subjects

Eisuke Miyauchi1, Masanori Tachikawa1*, Xavier Declèves2,3, Yasuo Uchida1, Jean-Luc Bouillot4, Christine Poitou5, Jean-Michel Oppert5, Stéphane Mouly2,6, Jean-François Bergmann2,6, Tetsuya Terasaki1, Jean-Michel Scherrmann2, Célia Lloret-Linares2,6

1

Membrane Transport and Drug Targeting Laboratory, Graduate School of Pharmaceutical

Sciences, Tohoku University, Sendai 980-8578, Japan 2

Inserm, UMR-S 1144 Université Paris Descartes-Paris Diderot, Variabilité de réponse aux

psychotropes, Paris F-75010, France 3

Assistance Publique-Hôpitaux de Paris, Hôpital Cochin, Pharmacokinetics and

Pharmacochemistry Unit, Paris F-75014, France 4

Assistance Publique-Hôpitaux de Paris, Hôpital Ambroise Paré, Université Versailles Saint

Quentin, Department of Surgery, Boulogne 92100, France 5

Assistance Publique-Hôpitaux de Paris, Groupe Hospitalier Pitié-Salpêtrière, Service de

Nutrition, Université Pierre et Marie Curie, Institut cardiométabolisme et nutrition (ICAN), Paris F-75013, France 6

Assistance Publique-Hôpitaux de Paris, Hôpital Lariboisière, Therapeutic Research Unit,

Department of Internal Medicine, Paris F-75010, France

1

ACS Paragon Plus Environment


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Running title: Quantitative protein atlas in jejunum of obese subjects

*Address all correspondence to: Associate Professor, Masanori Tachikawa, Ph.D., Membrane Transport and Drug Targeting Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan Tel: 81-22-795-6831; Fax: 81-22-795-6886 Email: [email protected]

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Abstract Protein expression levels of drug-metabolizing enzymes and transporters in human jejunal tissues excised from morbidly obese subjects during gastric bypass surgery were evaluated using quantitative targeted absolute proteomics. Protein expression levels of 15 cytochrome P450

(CYP)

enzymes,

10

UDP-glucuronosyltransferase

(UGT)

enzymes,

and

NADPH-P450 reductase (P450R) in microsomal fractions from 28 subjects and 49 transporters in plasma membrane fractions from 24 of the same subjects were determined using liquid chromatography-tandem mass spectrometry. Based on average values, UGT1A1, 2B15, and 2B17, SGLT1, and GLUT2 exhibited high expression levels (over 10 fmol/µg protein), though UGT2B15 expression was detected at a high level in only one subject. CYP2C9, 2D6, 3A5, UGT1A6, P450R, ABCG2, GLUT5, PEPT1, MCT1, 4F2 cell-surface antigen heavy chain (4F2hc), LAT2, OSTα and OSTβ showed intermediate levels (1–10 fmol/µg protein), and CYP1A1, 1A2, 1B1, 2C18, 2C19, 2J2, 3A7, 4A11, 51A1, and UGT1A3, 1A4, 1A8, 2B4, ABCC1, C4, C5, C6, and G8, TAUT, OATP2A1, 2B1, 3A1, and 4A1, OCTN1, CNT2, PCFT, MCT4, GLUT4 and SLC22A18 showed low levels (less than 1 fmol/µg protein). The greatest inter-individual difference (364-fold) was detected for UGT2B17. However, differences in expression levels of other quantified UGTs (except UGT2B15 and UGT2B17), CYPs (except CYP1A1 and CYP3A5), and P450R, and all quantified transporters, were within 10-fold. Expression levels of CYP1A2 and GLUT4 were significantly correlated with body-mass index. The levels of 4F2hc showed significant gender differences. Smokers showed increased levels of UGT1A1, and 1A3. These findings provide a basis for understanding the changes in molecular mechanisms of jejunal metabolism and transport, as well as their inter-individual variability, in morbidly obese patients.

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Key words Small intestine, morbid obesity, transporter, BCRP/ABCG2, OATP2B1, drug-metabolizing enzymes, CYP, UGT2B17

Abbreviations 4F2hc, 4F2 cell-surface antigen heavy chain; ABC, ATP-binding cassette; ASBT, apical sodium-dependent bile acid transporter; BCRP, breast cancer resistance protein; BMI, body-mass index; CFTR, cystic fibrosis transmembrane conductance regulator; CNT, concentrative nucleoside transporter; CYP, cytochrome P450; GLUT, glucose transporter member;

LAT,

large

neutral

amino

acids

transporter;

LC–MS/MS,

liquid

chromatography–tandem mass spectrometry; LOQ, limit of quantification; MATE, multidrug and toxin extrusion protein; MCT, monocarboxylate transporter; MDR, multidrug resistance protein; MRP, multidrug resistance-associated protein; OAT, organic anion transporter; OATP, organic anion-transporting polypeptide; OCT, organic cation transporter; OCTN, organic cation/carnitine transporter; OST, organic solute transporter; P450R, NADPH-P450 reductase; PBPK, physiologically based pharmacokinetics; PCFT, proton-coupled folate transporter; PEPT, peptide transporter; P-gp, P-glycoprotein; PMAT, plasma membrane monoamine transporter; QTAP, quantitative targeted absolute proteomics; RYGB, Roux-en-Y gastric bypass surgery; SD, standard deviation; SEM, standard error of mean; SGLT, sodium/glucose cotransporter; SLC, solute carrier; SLCO, solute carrier organic anion transporter family member; SRM/MRM, selected/multiple reaction monitoring; TAUT, sodium- and chloride-dependent taurine transporter; UGT,

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UDP-glucuronosyltransferase; ULQ, under the limit of quantification.

Abstract graphic

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Introduction The prevalence of obesity, defined as body-mass index (BMI) of over 30, has increased substantially worldwide over the last 30 years1. Compelling evidence has emerged of obesity-related changes in the pharmacokinetics of orally administered drugs2, e.g., increased oral bioavailability of midazolam3, and changes of mucosal permeability that would influence drug disposition due to perturbation of intestinal microbiota4. While most studies have focused on changes in liver metabolism2, the molecular mechanisms underlying changes of metabolism and transport processes in the small intestine of obese subjects, and their inter-individual differences remain largely unknown. A better understanding is important, because it could enable mechanism-based prediction of first-pass effects and drug-drug interaction of orally administered drugs in obese sub-populations by using physiologically based pharmacokinetic (PBPK) models. It might also open up opportunities to intervene in the mechanisms of progression of obesity, and to identify enzymes and/or transporters that could be targeted to improve intestinal absorption of various types of drugs or prodrugs. The small intestine possesses a variety of metabolizing enzymes, such as cytochrome P450 (CYP)5, 6 and UDP-glucuronosyltransferase (UGT), 7 and transporters2, 8, 9, which may either hinder or facilitate oral absorption of drugs and endogenous nutrients. As the jejunum is one of the major intestinal absorption sites, we considered that it would be useful to understand the activities of metabolizing enzymes and transporters in the small intestine of morbidly obese subjects. In this connection, it has been found that protein expression levels of organic cation/carnitine transporter 1 (OCTN1) and multidrug resistance-associated

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protein 1 (MRP1)/ATP-binding cassette (ABC) transporter C1 (ABCC1) are well correlated with the transport activities in primary cultured human tracheal, bronchial, and alveolar epithelial cells10. On the other hand, we have shown that mRNA expression levels of CYP enzymes, except for CYP3A4, are poorly correlated to metabolizing activity, and further, there is no correlation between mRNA and protein expression levels of transporters in human liver11. It is also established that there are significant species differences in first-pass effects among humans, dogs and rodents12. Therefore, we considered that direct protein quantification of metabolizing enzymes and transporters in jejunal tissues of morbidly obese patients would be the most suitable approach for the present purpose. We

have

developed

liquid

chromatography–tandem

mass

spectrometry

(LC–MS/MS)-based quantitative-targeted absolute proteomics (QTAP)13 and applied it for quantification of CYP and UGT isoforms, and ABC and solute carrier (SLC) transporters in human liver tissues11, blood-brain barrier-forming human brain microvessels14 and blood-cerebrospinal barrier-forming choroid plexus tissues15 in order to understand the physiological roles and pharmacokinetics of these functional proteins in humans in terms of protein expression amounts16. In addition, we have determined the individual expression levels of CYP3A4, UGT2B7, P-glycoprotein (P-gp)/MDR1/ABCB1, MRP2/ABCC2, and MRP3/ABCC3, which influence the pharmacokinetics of oral morphine and its glucuronides, in the jejunum of 26-28 morbidly obese subjects; we found, for example, that UGT2B7 amount is associated with the area under the blood-concentration curve of morphine17. Building on that work, we considered that establishing a quantitative protein atlas in jejunal tissues of obese patients would enable us to understand how the molecular

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mechanisms of intestinal absorption are altered in obese patients, as well as the extent of the inter-individual differences. Thus, the purpose of the present study was to quantify protein expression levels of metabolizing enzymes such as CYP and UGT isoforms besides CYP3A4, and UGT2B7, and NADPH-cytochrome P450 reductase (P450R), as well as nutrients and drugs transporters besides P-gp, MRP2 and MRP3, in jejunal tissue excised from morbidly obese subjects undergoing bariatric surgery by means of QTAP.

Experimental Section Patients: The patients (5 males and 23 females) were subjected to laparoscopic Roux-en-Y gastric bypass surgery (RYGB) (Table S1). Decision for operation was made by a multidisciplinary team including physicians, surgeons, anesthetists, dieticians, nurses, and psychologists according to the guidelines for the management of obese patients issued by consensus conferences18. Subjects with diabetes, renal or hepatic impairment, and untreated obstructive sleep apnea syndrome were excluded from this study. All subjects gave written informed consent to participate in the study. The protocol was approved by the regional ethics committee of Paris, France (CPP Ile de France I) and registered at ClinicalTrials.gov, with an EudraCT number 2009-010670-38. The QTAP-based protein quantification was approved by the ethics committee of the Graduate School of Pharmaceutical Sciences, Tohoku University, Japan. Intestinal Tissues: Jejunal tissues were obtained from patients undergoing RYGB surgery, which was performed by the same technique in the same department of surgery19. A

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fragment of jejunal mucosa, derived from a site about 2 meters from the usual gastroduodenal junction, and considered as surgical waste, was flash-frozen in liquid nitrogen immediately after resection. The jejunal tissues were stored for two to four years at -80ºC, and the frozen tissues were thawed only once at the time of the sample preparation. The sample preparation procedures, including membrane protein fraction preparation and proteolysis followed by LC-MS/MS analysis, were performed independently, but at the same time, for the present study and the previous study in which CYP3A4, UGT2B7, P-gp, MRP2, and MRP3 were analyzed17. Therefore, there would be no temporal bias between the data reported in the present and previous studies17. Preparation of Microsomal and Plasma Membrane Fractions: Frozen jejunal tissues were homogenized using a Potter-Elvehjem homogenizer in buffer A containing (in mM) 10 Tris–HCl (pH 7.4), 10 NaCl, 1.5 MgCl2, 1 phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO). The homogenates were additionally subjected to nitrogen cavitation (450 psi for 15 min at 4°C) in buffer A. The obtained homogenates were centrifuged at 10,000 g for 10 min at 4°C twice, and the supernatants were ultracentrifuged at 100,000g for 40 min at 4°C. Each obtained pellet was suspended in buffer B containing (in mM) 10 Tris-HCl buffer (pH 7.4) and 250 sucrose, and a part of the solution was stored at -80°C as microsomal fraction. The remaining solution was layered on top of a 38% (w/v) sucrose solution and centrifuged at 100,000g for 40 min at 4°C. The turbid layer at the interface was recovered, suspended in buffer B, and centrifuged at 100,000g for 40 min at 4°C. The plasma membrane fraction was obtained from the resulting pellet, which was suspended in buffer B. Protein concentrations

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were determined by the Lowry method using the DC protein assay reagent (Bio-Rad Laboratories, Hercules, CA). A total of 28 jejunal microsomal fractions of morbidly obese patients (5 males and 23 females) were obtained. Only 24 jejunal plasma membrane fractions of morbidly obese patients (5 males and 19 females) could be obtained, because the tissue volumes of 4 subjects were too small. LC-MS/MS-based QTAP analysis: Simultaneous protein quantitation of target molecules was performed by using nanoLC-MS/MS with multiplexed selected/multiple reaction monitoring (SRM/MRM) as described previously15. Protein expression levels were determined as the absolute amounts of trypsin-generated specific target peptides whose sequences were selected based on in silico selection criteria13. The SRM/MRM transitions for the quantification of each peptide were set as shown in supplemental Table S4. Preparation of trypsin- and lysylendopeptidase-treated samples, LC separation of peptides, and detection/quantification of the target peptides were carried out as we reported previously15 with several modifications as follows. In nanoLC separation, linear gradients of 0 to 40% acetonitrile in 0.1% formic acid for 40 min were applied to elute the peptides at a flow rate of 300 nL/min for nanoLC. The individual expression amount of each peptide was determined as an average of two to four SRM transitions from one sample in one analysis (Tables S2 and S3). The amount of PEPT1 was determined from only one SRM transition due to the presence of high noise levels at the other transitions (Table S3). The expression level of each molecule in all subjects was calculated as the mean±S.D. of the average of individual expression amounts for each molecule (Tables 1 and 2). The absolute expression amount of UGT1A8 was calculated from the quantitative data obtained for a

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peptide generated from both UGT1A8 and UGT1A9 by subtracting the value obtained for a peptide that is specific for UGT1A9. In cases where no signal peak was detected, the amount of peptide in the sample was defined as under the limit of quantification. The limit of quantification (fmol/µg protein) was calculated as the peptide amount expected to give a peak area count of 1000 in the chromatogram of samples. When the calibration curve was obtained with eqn 1, the peptide amount (fmol) expected to give a peak area count of 1000 (ATarget eq1000) was calculated from the peak area (counts) of internal standard peptide in the samples (PAIS in sample) and the values of slope and intercept in eqn 1, according to eqn 2. Then, the limit of quantification was obtained with eqn 3 by dividing ATarget eq1000 by the total protein amount (µg protein) of the sample analyzed (Asample). PASt in Authentic/PAIS in Authentic = Slope x ASt in Authentic + Intercept

(1)

ATarget eq1000 = (1000 counts/PAIS in sample – Intercept)/Slope

(2)

LOQ = ATarget eq1000/ASample

(3)

where PASt in Authentic and PAIS in Authentic are the peak areas (counts) of standard peptide and internal standard peptide in authentic samples, respectively, and ASt in Authentic is the amount (fmol) of standard peptide in authentic samples. Statistical analyses: Single regression analysis of protein expression levels, and BMI or age was performed by Excel software. Mann-Whitney's U test, an unpaired non-parametric method, was used to determine the statistical significance of differences between two groups. A p-value of less than 0.05 was considered as statistically significant.

Results

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Protein expression profiles of cytochrome P450, UDP-glucuronosyltransferase and NADPH-cytochrome P450 reductase enzymes in human jejunal microsomal fractions A total of 28 jejunal microsomal fractions were analyzed to examine inter-individual and inter-isoform variations in the expression levels of 15 CYP and 10 UGT isoforms, NADPH-cytochrome P450 reductase (P450R), epithelial marker villin-1, and epithelial plasma membrane marker Na+/K+-ATPase. Table 1 and Table S2 summarize the absolute expression amounts of 12 CYP isoforms, 8 UGT isoforms, P450R, villin-1, and. Na+/K+-ATPase, all of which were detected in at least one subject. The expression levels of CYP3A4 and UGT2B7 obtained in the previous study17 are included in Table 1 for comparison. The differences in the average expression levels of villin-1 and Na+/K+-ATPase were within 3.1- and 2.3-fold, respectively. UGT isoforms 1A1, 2B15, and 2B17 exhibited high expression levels of more than 11 fmol/µg protein on average. CYP isoforms 2C9, 2D6, and 3A5, UGT1A6, and P450R showed intermediate expression levels of 1.29 to 7.22 fmol/µg protein, while the remaining 9 CYP isoforms, 1A1, 1A2, 1B1, 2C18, 2C19, 2J2, 3A7, 4A11, and 51A1, and 4 UGT isoforms, 1A3, 1A4, 1A8, 2B4, showed low expression levels (less than 0.838 fmol/µg protein). CYP isoforms 2B6, 2C8, and 2E1, and UGT isoforms 1A9 and 2B10 were below the limit of quantification in all 28 samples analyzed. The greatest inter-individual variation (364-fold) was detected for UGT2B17, which showed expression levels of 88.6-to-0.244 fmol/µg protein among 27 quantified subjects. Two subjects had very low UGT2B17 expression levels of less than 0.224 fmol/µg protein. One of these two subjects showed a high expression level of UGT2B15 (25.2 fmol/µg

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protein). Since the expression levels of UGT1A1 (8.91 and 9.09 fmol/µg protein) in these two subjects were close to the average value of 11.0 fmol/µg protein, the possibility of low purity of the microsomal fractions of these subjects can be excluded. The expression levels of CYP1A1 and CYP3A5 varied by 34.5- and 19.9-fold among 15 and 28 subjects, respectively. Expression levels of the remaining isoforms of CYPs and UGTs (except for UGT2B15 and UGT2B17), and P450R varied by less than 10-fold among individuals, when values below the limit of quantification were excluded.

Protein expression profiles of ABC and SLC transporters in plasma membrane fraction of human jejunal tissues A total of 24 jejunal plasma membrane fractions were analyzed to examine inter-individual variations in the expression levels of 49 transporters, epithelial marker villin-1 and epithelial plasma membrane marker Na+/K+-ATPase. Table 2 and Table S3 summarize the absolute expression amounts of 6 ABC transporters, 20 SLC transporters, villin-1, and Na+/K+-ATPase, all of which were detected in at least 11 subjects. The expression levels of P-gp, MRP2, and MRP3 obtained in the previous study17 are included in Table 2 for comparison. The differences in the average expression levels of villin-1 and Na+/K+-ATPase were 2.6- and 2.3-fold, respectively. The average expression level of Na+/K+-ATPase in plasma membrane fraction (Table 2) was greater than that in microsomal fraction (Table 1). The sugar transporters SGLT1 and GLUT2 showed high average expression levels of more than 22.7 fmol/µg protein. The ABC transporter ABCG2 and 7 SLC transporters GLUT5, PEPT1, MCT1, 4F2hc, LAT2, OSTα and OSTβ exhibited

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intermediate expression levels (1.25 to 6.45 fmol/µg protein). Five ABC transporters (ABCG8, ABCC1, ABCC4, ABCC5 and ABCC6) and 7 SLC transporters (TAUT, OATP2B1, OCTN1, CNT2, PCFT, MCT4, GLUT4, OATP2A1, OATP3A1, OATP4A1, and SLC22A18) were classified as showing low-level expression (less than 1 fmol/µg protein). In contrast, 3 ABC transporters (ABCB4, ABCC7 and ABCG5) and 20 SLC transporters, including ASBT, OATP1A2, OCT1, OCT3, and LAT1, were below the limit of quantification in all 24 samples analyzed. The inter-individual differences in expression levels of quantifiable transporters (Table 2) were within 10-fold. The expression levels of ABCG2, PEPT1, CNT2, MRP4, MRP5, OATP3A1, and SLC22A18 exhibited over 5-fold differences.

Effect of body-mass index (BMI), gender, and smoking on the expression levels of CYPs, UGTs, P450R and transporters. We found a correlation (R2 = 0.577) between the expression levels of BMI and CYP1A2 in 9 subjects (Fig. 1A); the CYP1A2 levels of the other 19 subjects were under the limit of quantification. There was a positive correlation (R2 = 0.538) between the expression levels of BMI and GLUT4 in 11 subjects (Fig. 1B); GLUT4 levels of the other 13 subjects were under the limit of quantification. The expression levels of 4F2hc showed a significant gender difference between the 5 males and 19 females (Fig. 1C). A significant difference between 3 smokers and 25 non-smokers was found in the expression levels of UGT1A1 (Fig. 1D), and UGT1A3 (Fig. 1E). No significant correlation with the coefficient of determination R2 >0.5 was detected between BMI and the other CYPs, UGTs, P450R and

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Figure legends Figure 1. Effect of body-mass index (BMI), gender, and smoking on the expression levels of CYP1A2 (A), GLUT4 (B), 4F2hc (C), UGT1A1 (D), and UGT1A3 (E). (A and B) Correlation between BMI and the expression levels of CYP1A2 (A) (n=9), and GLUT4 (B) (n=11) in obese subjects. (C) Tukey’s box plot for gender difference in the expression levels of 4F2hc (n=5 males and 19 females). (D and E) Tukey’s box plot for the effect of smoking on the expression levels of UGT1A1 (D), and UGT1A3 (E) (n=3 smokers and 25 non-smokers). *p

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