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Pulmonary, gastrointestinal and urogenital pharmacology Orlistat limits cholesterol intestinal absorption by Niemann-pick C1- like 1 (NPC1L1) inhibition Saeed Alqahtani, Hisham Qosa, Brian Primeaux, Amal Kaddoumi n Department of Basic Pharmaceutical Sciences, School of Pharmacy, University of Louisiana at Monroe, Monroe, LA 71201, USA a r t i c l e i n f o Article history: Received 4 March 2015 Received in revised form 26 May 2015 Accepted 29 May 2015 Available online 3 June 2015 Keywords: Orlistat Cholesterol NPC1L1 abstract The known mechanism by which orlistat decreases the absorption of dietary cholesterol is by inhibition of intestinal lipases. The aim of this study was to investigate the ability of orlistat to limit cholesterol absorption by inhibition of the cholesterol transport protein Niemann-Pick C1-like 1 (NPC1L1) as another mechanism of action. In situ rat intestinal perfusion studies were conducted to study the effect of orlistat on jejunal cholesterol absorption. Inhibition kinetic parameters were calculated from in vitro inhibition studies using Caco2 and NPC1L1 transfected cell lines. The in situ studies demonstrated that intestinal perfusion of orlistat (100 mM) was able to reduce cholesterol absorption by three-fold when compared to control (i.e. in the absence of orlistat, Po0.01). In vitro studies using Caco2 cells demonstrated orlistat to reduce the cellular uptake of cholesterol by 30%. Additionally, orlistat reduced the cellular uptake of cholesterol in dose dependent manner in NPC1L1 transfected cell line with an IC50¼1.2 mM. Lineweaver– Burk plot indicated a noncompetitive inhibition of NPC1L1 by orlistat. Beside the already established mechanism by which orlistat reduces the absorption of cholesterol, we demonstrated for the ﬁ rst time that orlistat limits cholesterol absorption by the inhibition of NPC1L1 transport protein. & 2015 Elsevier B.V. All rights reserved. 1. Introduction The transport of cholesterol across the apical membrane of en- terocytes requires speci ﬁ c transport proteins rather than passive dif- fusion. In 2004, Altmann and colleagues identi ﬁ ed Niemann-Pick C1- like 1 (NPC1L1) as the responsible protein for cholesterol absorption across the intestinal membrane (Altmann et al., 2004). In humans, NPC1L1 is abundantly expressed in the small intestine and in the liver (Jia et al., 2011; Yu et al., 2006). The intracellular localization of NPC1L1, however, remains controversial. Several studies have shown NPC1L1 localization in the apical cell membrane of the small in- testine’s enterocytes acting as a transporter (Altmann et al., 2004; Davis and Altmann, 2009), while others have reported its localization predominantly in the perinuclear region functioning as a receptor (Davies et al., 2005; Ge et al., 2008). Nevertheless, NPC1L1 plays sig- ni ﬁ cant role in cholesterol transport and has been utilized as a ther- apeutic target to control cholesterol blood levels. An example of such therapeutics is ezetimibe, a novel inhibitor of cholesterol intestinal absorption, which limits the absorption of dietary and biliary choles- terol by inhibiting NPC1L1 cholesterol transport across the en- terocytes’ apical membrane (Van Heek et al., 1997). Orlistat, also known as tetrahydrolipstatin (Fig. 1), is a re- versible inhibitor of gastric and pancreatic lipases, the enzymes that break-down intestinal triglycerides (TGs) (Curran and Scott, 2004; Henness and Perry, 2006). Orlistat works locally in the lu- men of the stomach and small intestine by binding covalently to the serine active site in the lipase enzyme, preventing it from hydrolyzing dietary TGs into absorbable free fatty acids (Henness and Perry, 2006; Xenical, s2013). Undigested TGs are then excreted in feces, resulting in an inhibition of dietary fat absorption by 30% at the approved dosage (Xenicals, 2013). In addition, orlistat is known to decrease dietary cholesterol absorption by inhibition of intestinal lipases (Mittendorfer et al., 2001). Orlistat’s absorption and metabolism are minimal with 83% of the drug is primarily eliminated intact in the feces (Zhi et al., 1995, 1996). The effect of orlistat on cholesterol levels in obese patients with or without hypercholesterolemia compared to placebo has been studied previously (Guy-Grand et al., 2004; Lucas et al., 2003; Mittendorfer et al., 2001). Patients’ treatment with orlistat re- sulted in a signi ﬁ cant reduction in low-density lipoprotein (LDL)- cholesterol and TGs levels when compared to patients receiving placebo (Guy-Grand et al., 2004; Lucas et al., 2003; Mittendorfer et al., 2001), an effect that could not be explained only by weight loss. Thus, in the present study we hypothesized that in addition to intestinal lipases inhibition, the cholesterol-lowering property of orlistat could be related to inhibition of the cholesterol trans- port protein NPC1L1. In order to test this hypothesis, we aimed Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ejphar European Journal of Pharmacology http://dx.doi.org/10.1016/j.ejphar.2015.05.060 0014-2999/& 2015 Elsevier B.V. All rights reserved. n Correspondence to: School of Pharmacy, University of Louisiana at Monroe, 1800 Bienville Dr., Monroe, LA 71201, USA. Tel.: þ 1 318 342 1460; fax: þ1 318 342 1737. E-mail address: firstname.lastname@example.org (A. Kaddoumi). European Journal of Pharmacology 762 (2015) 263–269 ﬁ rst to investigate the effect of orlistat perfusion on cholesterol intestinal absorption, in the absence of lipases, using the in situ rat intestinal perfusion technique, and second to mechanistically in- vestigate orlistat inhibitory effect on NPC1L1 using in vitro studies with Caco2 and NPC1L1 transfected cell lines. 2. Materials and methods 2.1. Materials Orlistat ((2S)-1-[(2S,3S)-3-hexyl-4-oxooxetan-2-yl]tridecan-2- yl (2S)-2-formamido-4-methylpentanoate) was purchased from Sigma Aldrich (St Louis, MO). [14C] Cholesterol was purchased from PerkinElmer (Boston, MA). Sodium taurocholate, phosphati- dylcholine and oleic acid were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Ezetimibe was generously provided by Scher- ing Plough (Kenilworth, NJ). McArdle RH7777 rat hepatoma cells stably expressing the human NPC1L1-EGFP fusion protein (re- ferred to as L1-EGFP cells) and their respective control (EGFP cells) were generously provided by Dr. Liqing Yu (Department of Pa- thology, Wake Forest University School of Medicine, North Car- olina). Caco2 cells, originated from human colorectal adenocarci- noma, and supplies for cell culture were obtained from the American Type Cell Culture Collection (ATCC; Manassas, VA). Other chemicals and reagents were obtained from VWR (West Chester, PA). 2.2. Animals Male Sprague-Dawley rats weighing 260–300 g were acquired from Harlan Laboratories (Houston, TX). All animal experiments were approved by the Institutional Animal Care and Use Com- mittee (IACUC) of the University of Louisiana at Monroe and all surgical and treatment procedures were consistent with the In- stitutional Animal Care and Use Committee policies and proce- dures. The rats were maintained on a 12 h light/dark cycle before the study and were fasted for 12–18 h with water ad libitum before each experiment. 2.3. In situ rat intestinal perfusion model This experiment was conducted as described previously (Abuasal et al., 2010). On the day of study, after overnight fasting, rats were anesthetized by intraperitoneal injection of 1 g/kg ur- ethane. The small intestine was exposed by midline incision. Ap- proximately 15 cm of the jejunum was externalized, ﬂ ushed with warm normal saline to remove intestinal contents, and then can- nulated with glass cannulas inserted at the inlet and the outlet of the segment and were secured by ligation with silk suture. The inlet tubing of the segment was connected to a 30 ml syringe that was placed in an infusion pump (Harvard Apparatus Inc., Holliston, MA). Animals, perfusion solutions, and pump were enclosed in a thermostatically controlled Plexiglas chamber (Widgett Scienti ﬁ c Inc., Baton Rouge, LA) set at 30 °C. The perfusate was pumped through the lumen at 0.14 ml/min ﬂ ow rate. The perfusate solution consisted of [14C] cholesterol (0.02 mCi/ml) in the absence or pre- sence of orlistat (100 mM), n¼3 rats per group, prepared as mixed micelles in 1.6 g/l sodium taurocholate and 0.575 g/l phosphati- dylcholine in phosphate buffer (pH 6.5). The ﬁ rst 40 min pre- steady-state outlet perfusate was discarded, as the data re- presented the stabilization period prior to reaching steady state. The outlet perfusate was then collected in vials at 10 min intervals for 100 min. The samples were mixed with scintillation cocktail and [14C] cholesterol was then analyzed using Wallac 1414 Win- Spectral Liquid Scintillation Counter (PerkinElmer, MA). The ef- fective permeability (Peff) of cholesterol across the rat intestine was calculated based on its loss from the perfusate according to the equation (Abuasal et al., 2010): P Q C C rL ln / 2eff t 0 () π = −× where Q is the perfusate ﬂ ow rate through the segment (0.14 ml/ min), r is the radius of the intestinal lumen (0.2 cm), L is the length of the perfused segment (15 cm), Co is cholesterol concentration at the start of perfusion (from the entry tubing) and Ct is the steady state cholesterol concentration exiting the perfused intestinal segment. 2.4. Cholesterol mixed micelles (MM) preparation Cholesterol mixed micelles (MM) was prepared as described previously (Alqahtani et al., 2013a). In brief, [14C] cholesterol di- luted in ethanol, phosphatidylcholine dissolved in methanol, taurocholate dissolved in 96% ethanol, and oleic acid diluted in methanol were mixed, and then evaporated to dryness under ni- trogen gas. A transport buffer (4 mM KCl, 141 mM NaCl, 1 mM MgSO4,10 mM glucose, 10 mM HEPES, and 2.8 mM CaCl2 adjusted to pH 7.4) was then added to prepare the medium for cellular uptake experiments. Final concentrations were: phosphati- dylcholine 150 mM, taurocholate 300 mM, oleic acid 500 mM, and the required [14C] cholesterol concentrations in each experiment. The micellar solution was thoroughly vortexed and stirred at 37 °C for 30 min. 2.5. Cell culture Caco2 cells, and McArdle-RH7777 rat hepatoma cells stably expressing EGFP (enhanced green ﬂ uorescent protein) (EGFP cells) or those expressing an NPC1L1-EGFP fusion protein (L1-EGFP cells) were used in this study at passage numbers ranging from 40 to 60 passages for Caco2 cells, and 40–50 passages for the hepatoma cells. NPC1L1 transfected cells has been fully characterized for increased expression of NPC1L1 and enhanced cholesterol uptake as a result of transfection compared to mock cells (Brown et al., 2007; Yu et al., 2006). For normal growth, Caco2, L1-EGFP, and EGFP cells were cultured in Dulbecco’s Modi ﬁ ed Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2.5% antibiotics (10,000 I.U. penicillin and 10 mg streptomycin per ml) in a humidi ﬁ ed incubator with 5% CO2 at 37 °C. Cells were cultured in 75 cm2 ﬂ asks at a density of 1106 cells/ ﬂ ask and were harvested at 90% con ﬂ uence with trypsin–EDTA. Cells were seeded onto a 48-well plate at a density of 50,000 cells/well. When con ﬂ uent, uptake studies were performed as described below. All in-vitro studies were conducted at minimum in triplicate independent assays, and each assay was performed in quadruplicate. Fig. 1. Chemical structure of orlistat. S. Alqahtani et al. / European Journal of Pharmacology 762 (2015) 263–269264 2.6. [14C] Cholesterol uptake and NPC1L1 inhibition studies Time-dependent inhibition studies with orlistat were con- ducted using Caco2 cells, an in vitro model of absorptive en- terocytes. [14C] cholesterol prepared as MM was diluted in trans- port buffer to prepare 0.02 mCi/ml ﬁ nal concentration. Two hun- dred microliters from the formulation was added to the cells and incubated for 0, 15, 30, 60, 120 min in absence or presence of 50 mM of orlistat. At the end of the incubation time, cells were washed and lysed with lysis buffer before analysis. For con- centration-dependent inhibition studies, [14C] cholesterol MM was diluted in transport buffer to prepare 0.02 mCi/ml of [14C] choles- terol in the presence of different concentrations of orlistat ranging from 2 to 100 mM. Two hundred microliters from each con- centration were added to the cells and incubated for 60 min at 5% CO2 at 37 °C. To determine cholesterol uptake by NPC1L1 transport protein, we conducted uptake studies using EGFP and L1-EGFP cell lines. [14C] cholesterol prepared as MM was diluted in transport buffer to prepare different concentration ranging from 0.1 to 0.8 nM of [14C] cholesterol. Two hundred microliters from each concentration were added to EGFP and L1-EGFP cells and incubated for 60 min. Inhibition studies with orlistat and ezetimibe (as a positive con- trol) were conducted in EGFP and L1-EGFP cells. [14C] Cholesterol (0.02 mCi/ml) was added to the cells in absence or presence of different concentrations of orlistat or ezetimibe in the range of 0.1–100 mM. At the end of uptake studies, cells were washed twice with cold PBS and 100 ml of the lysis buffer was added. Twenty ﬁ ve micro- liters aliquots from the cell lysate were collected at the end of incubation period. After mixing samples with scintillation cocktail, [14C] cholesterol dpm was measured using Wallac beta Counter. Concentrations of [14C] cholesterol in the samples were calculated in dpm/ml and then converted to pmol/mg protein. 2.7. Effect of 48 h treatment with orlistat on NPC1L1 activity To study the effect of orlistat treatment on NPC1L1 activity, L1- EGFP cells seeded in 48-well plate were treated with 10 or 100 mM orlistat. Forty eight hours later, cells were washed three times with PBS followed by treatment with 0.02 mCi/ml [14C] cholesterol for 60 min. Then, intracellular levels of radiolabeled cholesterol were measured. For normalization, the protein concentration of parallel cultures was determined using the BCA colorimetric assay. 2.8. Western blot analysis The analysis of NPC1L1 expression in EGFP and L1-EGFP cells treated with different concentrations of orlistat was performed as reported previously (Abuasal et al., 2010). In brief, 16 mg of protein extracts from cells lysate was resolved using 7.5% SDS-poly- acrylamide gel electrophoresis and transferred electrophoretically onto a nitrocellulose membrane. After blotting, the membrane was blocked using 2% bovine serum albumin in PBS. To detect NPC1L1 (175 kDa) and β-actin (46 kDa), used as a normalizing protein, the membrane was cut at molecular weight of 75 kDa into two parts. The membrane parts were then separately immunoblotted with either NPC1L1 rabbit polyclonal IgG primary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or β-actin (C-11) primary antibody (Santa Cruz Biotechnology, Inc.) at 1:200 and 1:3000 dilutions, respectively, and incubated overnight at 4 °C. For protein detection, the membranes were separately incubated with sec- ondary anti-rabbit IgG antibody for NPC1L1 and anti-goat IgG antibody for β-actin, both labeled with horseradish peroxidase, each at a 1:5000 dilution. The blots were developed using Pierce ECL Western Blotting Substrate Detection Kit (Thermo Fisher Scienti ﬁ c, Waltham, MA). Quantitative analysis of the im- munoblots was performed using LI-COR C-DiGits Blot Scanner (LI- COR Biosciences; Lincoln, NE) and band intensities were measured by densitometric analysis. 2.9. Immunoﬂuorescence microscopy For imaging studies, EGFP and L1-EGFP cells were plated in 8-well chambered glass slides (LAB-Tek, Nalge Nunc, NY) at 10,000 cells/cm2, and allowed to grow overnight. Cells were in- cubated with 10 and 100 mM of orlistat for 48 h. At the end of the incubation periods, cells were washed and ﬁ xed with 4% for- maldehyde for 5 min on ice. Cells were mounted with DAPI. Ima- ges for NPC1L1 were captured using Nikon inverted microscope Eclipse Ti-s (Nikon Instruments Inc; Melville, NY). 2.10. Statistical analysis GraphPad Prism, version 6.00 (GraphPad Software, San Diego, CA, USA) was used to perform all statistical analysis. The experi- mental results were statistically analyzed for signi ﬁ cant difference using two-tailed Student’s t-test for 2 two groups, and one-way analysis of variance (ANOVA) for more than two groups’ analysis. Vmax, KM, and IC50 were determined from nonlinear regression of the concentration versus cellular uptake curves by GraphPad Prism. The results were presented as mean7S.D. All in-vitro ex- periments were performed at minimum in triplicate independent assays, and each assay was performed in quadruplicate; and the in situ perfusion experiments were achieved by using n¼3 per group. P values o0.05 was considered statistically signi ﬁ cant. 3. Results 3.1. In situ rat intestinal perfusion studies Results from in situ studies demonstrated that the co-perfusion of 100 mM orlistat with [14C] cholesterol signi ﬁ cantly reduced the permeability (Peff ) of [ 14C] cholesterol by 3-fold from 4.810570.9105 cm/s in the absence of orlistat to 1.7 10570.6105 cm/s with orlistat (n¼3 per group, Po0.01; Fig. 2). Fig. 2. Effective permeability (Peff) of [14C] cholesterol using in situ intestinal per- fusion in presence and absence of 100 mM of orlistat (n¼3 per group). Each value represents the mean7S.D. ** indicates signi ﬁ cant difference compared to control (Po0.01). S. Alqahtani et al. / European Journal of Pharmacology 762 (2015) 263–269 265 3.2. Cholesterol uptake inhibition by orlistat in Caco2 cells Comparative effects of incubation time (15–120 min) on cel- lular uptake of [14C] cholesterol prepared as MM with or without 50 mM of orlistat are presented in Fig. 3A. The results showed signi ﬁ cant reduction in the cellular uptake of [14C] cholesterol in presence of orlistat at all-time points (Po0.05). Additionally, the results showed that the cellular uptake of [14C] cholesterol in- creased with time up to 2 h. Based on these results, all consequent uptake studies were performed at 1 h (in the linear range). Results from concentration dependent inhibition studies with orlistat on the cellular uptake of [14C] cholesterol following 1 h of treatment are presented in Fig. 3B. As illustrated in this ﬁ gure, there were no signi ﬁ cant changes in the cellular uptake of [14C] cholesterol in the presence of orlistat up to 10 mM( P40.05). The inhibitory effect of orlistat started at concentrations Z20 mM in a concentration dependent manner. The cellular uptake of [14C] cholesterol was signi ﬁ cantly lower in the presence of 20, 50, and 100 mM of orlistat with 17–30% reduction (Po0.05) compared to control cells in the absence of orlistat. 3.3. Kinetics of NPC1L1 inhibition by orlistat To con ﬁ rm that the inhibitory effect of orlistat on the cellular uptake of [14C] cholesterol is via NPC1L1, inhibition studies were conducted using EGFP and L1-EGFP cells. The net uptake of [14C] cholesterol by NPC1L1 was calculated as the difference between total cellular uptake of [14C] cholesterol by NPC1L1 transfected cells (L1-EGFP cells) and the respective vector control cells (EGFP cells). As illustrated in Fig. 4A, the net uptake of [14C] cholesterol by NPC1L1 in the absence of orlistat demonstrated a saturable process with Vmax and KM values of 0.4 nmol/mg protein/60 min and 0.13 nM, respectively. The net cellular uptake of [14C] choles- terol was reduced signi ﬁ cantly in the presence of 50 mM of orlistat and ezetimibe (Po0.05); ezetimibe showed a greater inhibition (Fig. 4A). The KM values were similar in presence and absence of inhibitors, whereas, the Vmax values for the [14C] cholesterol up- take were reduced in presence of orlistat and ezetimibe to 0.23 and 0.18 nmol/mg protein/60 min, respectively. Additionally, ki- netic studies on the inhibition of [14C] cholesterol cellular uptake by orlistat and ezetimibe were further examined using Line- weaver–Burk double reciprocal plot. The inhibition mode of orli- stat and ezetimibe was similar and was determined as non- competitive by the Lineweaver–Burk double reciprocal plot of re- action velocity versus substrate concentration (Fig. 4B), in which the KM was not altered by inhibitor. Orlistat inhibited cholesterol cellular uptake by NPC1L1 in a concentration dependent manner with an IC50 of 1.2 μM(Fig. 5A). The cellular uptake of cholesterol reduced signi ﬁ cantly by 55% at the highest concentration when co-incubated with different con- centrations of orlistat ranging from 0.1 to 100μM. The inhibitory effect of ezetimibe on the cellular uptake of cholesterol was higher compared to orlistat with IC50 of 0.37μM(Fig. 5B). 3.4. Effect of prolonged exposure of orlistat for 48 h on the activity and expression of NPC1L1 To investigate the effect of prolonged exposure of cells for 48 h with orlistat on the activity of NPC1L1, we treated NPC1L1 trans- fected cells (L1-EGFP) with low (10μM) and high (100 μM) con- centrations of orlistat for 48 h. The cellular uptake of [14C] cho- lesterol following 10μM treatments for 48 h was not signi ﬁ cantly altered when compared to control treatment (P40.05). Whereas, for cells treated with 100μM orlistat, the cellular uptake of cho- lesterol was signi ﬁ cantly higher compared to the control. As illu- strated in Fig. 6, the cellular uptake of cholesterol after treatment with 100μM of orlistat for 48 h was 22% higher compared to the control, a modest effect but signi ﬁ cant (Po0.05). Furthermore, Fig. 3. (A) Time-dependent studies with or without orlistat (50 mM), and (B) concentration-dependent studies of orlistat (0–100 mM) on the cellular uptake of 0.02 mCi/ml [14C] cholesterol (in pmole/mg protein) in Caco2 cells. Each value represents the mean7S.D. (n¼3 independent experiments). * Po0.05, ** Po0.01 compared to control. Fig. 4. The mode of NPC1L1 cholesterol uptake inhibition by orlistat and ezetimibe in EGFP and L1-EGFP cells. The net uptake of cholesterol by NPC1L1 was calculated as the difference between cellular uptake of [14C] cholesterol by NPC1L1 transfected cells (L1-EGFP cells) and the respective vector control cells (EGFP cells). The mode of the inhibition was analyzed using Michaelis–Menten model (A), or the Lineweaver–Burk plot (B) in the presence or absence of orlistat and ezetimibe at 50 mM concentration. S. Alqahtani et al. / European Journal of Pharmacology 762 (2015) 263–269266 consistent with results obtained from the concentration-depen- dent studies the acute treatment with 100μM of orlistat caused 50% reduction in the cellular uptake of [14C] cholesterol (P40.001). To test whether the enhanced cholesterol uptake after 48 h treatment with orlistat is due to increase in expression level of NPC1L1 or due to enhanced traf ﬁ cking of NPC1L1 from cyto- plasm to the cell membrane, we measured the amount of NPC1L1 in EGFP and L1-EGFP cells after treatment with 10 and 100 μM of orlistat by Western blot analysis and ﬂ uorescence imaging. Wes- tern blot analysis results showed that the protein expression of NPC1L1 was not altered by orlistat at the concentrations tested in both transfected and mock cells (Fig. 7A, B). The ﬂ uorescence images for NPC1L1 showed, however, that L1-EGFP cells treatment for 48 h with 100 μM orlistat stimulated NPC1L1 traf ﬁ cking from the cytoplasm to the cell membrane, which could explain the signi ﬁ cant increase in cholesterol uptake after the treatment (Fig. 7C). The treatment of L1-EGFP cells with 10μM orlistat, however, did not show any alteration in NPC1L1 localization or expression and was similar to those observed with untreated control cells (Fig. 7C). 4. Discussion Orlistat is known to decrease the absorption of dietary TGs, an effect that has been reported to result from its inhibition of intestinal lipases and the subsequent reduction in fat digestion in recipients of the drug. In addition to its effect on reducing TGs, available clinical studies have shown orlistat to reduce dietary cholesterol absorption. Clinical studies in obese patients ac- knowledged the contribution of an additional, yet to be identi ﬁ ed, mechanism(s) to the decreased levels of plasma cholesterol. Erd- mann and colleagues have reported that orlistat prevents the di- gestion of approximately 30% of the ingested fat (Erdmann et al., 2004), which is associated with 15–25% reduction in cholesterol plasma levels (Erdmann et al., 2004; Mittendorfer et al., 2001). The authors of these studies concluded that orlistat lowers plasma cholesterol beyond its effect on weight loss associated with di- gestion inhibition. Thus, in the current work, we hypothesized that in addition to its inhibitory effect on intestinal lipases, orlistat decreases cholesterol absorption by NPC1L1 inhibition as another potential mechanism of action. To investigate this hypothesis, all studies were performed in lipase free assays to separate orlistat inhibitory effect on cholesterol absorption/uptake from that of li- pases inhibition. In situ studies were ﬁ rst performed to evaluate the effect of orlistat, prepared in phosphate buffer solution, on cholesterol intestinal absorption. Then, to speci ﬁ cally examine the inhibitory mechanism of orlistat on the uptake of cholesterol in vitro studies using Caco2 and NPC1L1 transfected cells were per- formed. In addition, in these studies cholesterol was added to the cells as mixed micelles to re ﬂ ect post-digestion process, produced by lipases as the end product. In the intestinal lumen, most of available cholesterol is derived from internal sources via the bile, whereas the diet contributes a relatively minor fraction (up to 2 g/ day vs. 0.4 g/day, respectively) (Cohen, 2008). Within the in- testine, biliary cholesterol mixes with dietary cholesterol in form of micelles and become readily available for uptake by enterocytes. In human tissues, NPC1L1 is predominantly expressed in the liver with detectable levels in lung, heart, brain, pancreas, and kidney, ranging in expression from about 0.5% to 3% of liver ex- pression (Davies et al., 2005). Whereas, in the small intestine, NPClL1 is expressed at 1–4% of its level in the liver with highest expression in the proximal segment (Davies et al., 2005). To our knowledge, our study is the ﬁ rst to discuss the relationship be- tween orlistat and NPC1L1. All previous studies that showed the effect of orlistat on cholesterol absorption have not discussed the molecular mechanism of this effect. Several models have been used to study the intestinal ab- sorption of compounds. One of the most reliable models is the in situ single-pass intestinal perfusion in rats, which provides a valuable tool to assess the role of transport proteins and the in- hibitory effect of drugs on transport proteins in a speci ﬁ c segment of the intestine (Alqahtani et al., 2013b). The digestion process of dietary fat begins in the stomach where gastric lipase is released (Lowe, 1997). Then, secreted pancreatic lipases by pancreatic Fig. 5. Concentration-dependent inhibition of cholesterol cellular uptake by (A) orlistat, and (B) ezetimibe in EGFP and L1-EGFP cells. The concentrations investigated were in the range 0.1–100 mM for both drugs. Symbols represent means7S.D. of 4 independent experiments. Fig. 6. Cellular uptake of 0.02 mCi/ml [14C] cholesterol in the presence or absence of orlistat. L1-EGFP cells were acutely treated with 100 mM or treated for 48 h with 10 and 100 mM of orlistat. Each value represents the mean7S.D. of 3 independent experiments. * Po0.05, ** Po0.01 compared to control. S. Alqahtani et al. / European Journal of Pharmacology 762 (2015) 263–269 267 acinar cells into the duodenum would complete fat digestion in the proximal small intestine (Lowe, 1997). To eliminate the role of these lipase enzymes and investigate secondary mechanisms, in the in situ perfusion study the jejunum of the small intestine was separated and ﬂ ushed very well with normal saline before starting the experiment. Orlistat was perfused at a concentration of 100 μM, which is lower than expected following ingestion of the clinically recommended therapeutic dose of 120 mg taken with 250 ml of water. This dose projects an initial upper intestinal concentration of about 1000μM. However, due to orlistat poor water solubility, it was examined at 100μM prepared as mixed micelles. The results demonstrated that orlistat signi ﬁ cantly re- duced the Peff of cholesterol by approximately 3-fold (Fig. 2), suggesting that the reduced absorption of cholesterol could be related to its uptake inhibition by the enterocytes and not to cholesterol digestion process. In Caco2 cells, inhibition studies with orlistat showed a sig- ni ﬁ cant reduction in cholesterol intracellular levels when com- pared to orlistat untreated cells, however, as Caco2 cells express endogenous NPC1L1 (Kumar et al., 2011), in addition to other proteins that may or may not contribute to cholesterol uptake, McArdle RH7777 rat hepatoma cells stably expressing hNPC1L1 was also used to speci ﬁ cally investigate orlistat inhibitory effect on hNPC1L1. This cell line was used for 3 reasons: (1) this hNPC1L1 transfected hepatoma cell line is very well characterized and widely used to study cholesterol transport (Abuasal et al., 2012; Brown et al., 2007); (2) it provides an excellent model to speci ﬁ - cally study orlistat inhibitory effect on NPC1L1, which is the ob- jective of this study; and 3) it is a valid model for mechanistic studies where the effect of other process, other than NPC1L1, that might contribute to cholesterol uptake can be omitted by per- forming similar studies in mock cells of same cell line to isolate the speci ﬁ c inhibitory effect of orlistat on NPC1L1 from other possible processes, which was successfully done and allowed us to estimate all kinetic parameters of NPC1L1 inhibition by orlistat. The cellular uptake of cholesterol by NPC1L1 was calculated as the difference in cholesterol uptake between the transfected and mock cells. Our ﬁ ndings demonstrated that the cellular uptake of cholesterol was signi ﬁ cantly reduced in the presence of 50 μM of orlistat and ezetimibe (as a positive control) with a greater inhibition caused by the later (Fig. 4A). The mode of inhibition of orlistat was ex- amined by measuring NPC1L1 uptake activity with different con- centrations of substrate using Lineweaver–Burk plot analysis. The results showed that like ezetimibe, orlistat inhibits NPC1L1 by a noncompetitive mechanism. The Vmax values for the cholesterol cellular uptake were signi ﬁ cantly reduced while the KM values were not altered by the inhibitors (Fig. 4B). In order to evaluate the inhibitory potential of orlistat on the cellular uptake we measured the cellular uptake of cholesterol in presence of increasing con- centrations of orlistat. The results demonstrated that the potency of orlistat as an inhibitor of NPC1L1 revealed an IC50 value of 1.2 μM which was 4-fold higher than that of ezetimibe with an IC50 of 0.37μM(Fig. 5). Although orlistat’s potency to inhibit NPC1L1 function is 20-fold lower than lipase inhibition, the ori- ginal mechanism that has an IC50 of approximately 60 nM (Bi- sogno et al., 2006), it demonstrates an additional mechanism of action by which orlistat could reduce the absorption of dietary cholesterol. This NPC1L1 inhibitory effect could be small and Protein:actinratio EGFP 0 µM EGFP 10 µM EGFP1 00 µM L1-EGFP0µM L1-EGFP 10 µM L1-EGFP 100 µM 0.0 0.1 0.2 0.3 0.4 L1-EGFP0µM L1-EGFP100 µML1-EGFP10µM Fig. 7. The effect of orlistat on the protein expression and localization of NPC1L1 in EGFP and L1-EGFP cells. (A) Western blot analysis of cellular NPC1L1 after treatment with orlistat at 10 and 100 mM for 48 h. The predicted molecular mass of the unmodi ﬁ ed NPC1L1-EGFP fusion protein is 175 kDa (145 kDa for NPC1L1 and 30 kDa for EGFP). (B) Densitometry analyses of the blots showed no signi ﬁ cant changes in the expression of NPC1L1 following orlistat treatment when compared to control. (C) Fluorescence images for L1-EGFP cells, untreated, and treated with 10 and 100 mM of orlistat for 48 h. S. Alqahtani et al. / European Journal of Pharmacology 762 (2015) 263–269268 secondary to lipases inhibition; however, as a mechanism con- tributing to limiting cholesterol absorption could be signi ﬁ cant especially for obese patients with co-morbidities such as hy- percholesterolemia and heart diseases. Findings from Western blot analysis demonstrated that cells treatment with orlistat for 48 h has no effect on the expression level of NPC1L1 protein (Fig. 7), suggesting that the relocation of NPC1L1 to the cell membrane could explain the signi ﬁ cant in- crease in cholesterol cellular uptake after the prolonged exposure of NPC1L1 transfected cells to 100μM of orlistat (Fig. 6). Visual analysis of the ﬂ uorescence microscopic images of L1-EGFP cells showed an increase in the expression of NPC1L1 at the cell membrane following treatment with 100μM of orlistat for 48 h (Fig. 7). This result in addition to the Western blot data con ﬁ rm that the prolonged treatment of cells with orlistat causes NPC1L1 traf ﬁ cking and relocation to the cell membrane, possibly due to reduced intracellular cholesterol after orlistat treatment. Further studies are required to investigate this observation. These ﬁ ndings, however, are in agreement with a previous study reported the relocation of NPC1L1 to the cell membrane after cholesterol de- pletion by methyl-β-cyclodextrin (Yu et al., 2006). This translo- cation was associated with an increase in cellular cholesterol up- take which was inhibited by ezetimibe (Yu et al., 2006). Collectively, results of this study demonstrated the ability of orlistat to inhibit cholesterol absorption by inhibiting NPC1L1. In clinical settings, orlistat is given with food 3 times a day; while this chronic treatment could increase NPC1L1 level at cell surface due to its relocation, the millimolar intestinal concentrations of orlistat is expected to inhibit the absorption of dietary cholesterol. This distinctive mechanism of inhibition of NPC1L1 by orlistat beside its already established mechanism makes the idea of using it to lower cholesterol levels in hypercholesterolemia very attrac- tive. These ﬁ ndings could broaden the therapeutic options for hypercholesterolemia. 5. Conclusions In summary, besides the already established mechanism by which orlistat reduces the absorption of cholesterol, we demon- strated for the ﬁ rst time that orlistat has the potential to reduce cholesterol absorption through the inhibition of NPC1L1 transport protein. Further investigations are required to identify the mode of binding to NPC1L1 and if any further chemical modi ﬁ cations will enhance its potency. Con ﬂ ict of interest The authors have no con ﬂ ict of interest to declare. References Abuasal, B., Sylvester, P.W., Kaddoumi, A., 2010. Intestinal absorption of gamma- tocotrienol is mediated by Niemann-Pick C1-like 1: in situ rat intestinal per- fusion studies. Drug Metab. Dispos. 38, 939–945. Abuasal, B.S., Qosa, H., Sylvester, P.W., Kaddoumi, A., 2012. 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