The Effect of Methylated Lithocholic Acid on Plasma Membrane Properties: The Difference with the Effect of Cholesterol

The Effect of Methylated Lithocholic Acid on Plasma Membrane Properties: The Difference with the Effect of Cholesterol | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Effect of Methylated Lithocholic Acid on Plasma Membrane Properties: The Difference with the Effect of Cholesterol Tomoyuki Iwasaki, Nobuyuki Endo, Yuta Nakayama, Toshiyuki Kamei, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1700469/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bile acids form micelles that are essential for absorbing dietary lipids. However, excessive amounts of bile acid micelles may disrupt the plasma membrane by removing phospholipids, resulting in cell death. We hypothesized that the bent geometrical structure of the steroid scaffold of bile acids decreases lipid order (similar to unsaturated phospholipids with cis double bonds), disrupting the plasma membrane. Here, lithocholic acid (LCA), a bile acid, was methylated to prevent micellization. Methylated lithocholic acid (Me-LCA) was mixed with a thin phase-separated lipid layer comprising 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl- sn -glycero-3-phosphocholine (DPPC), and cholesterol (Chol). Me-LCA was localized not in the gel phase but in the liquid crystalline phase and excess Me-LCA did not affect phase separation. Me-LCA was distributed in the plasma membrane and the organelle membranes. However, the Me-LCA did not affect the membrane properties, membrane fluidity, and hydrophobicity of liposomes composed of DOPC, DPPC and Chol, and it did not affect proliferation. These results suggest that the removal of phospholipids from the plasma membrane by bile acid micelles mainly contributes to cell death. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The plasma membrane is composed of several types of lipids. The localization of these lipids in the plasma membrane is regulated, and these lipids play key biological roles. For example, the localization of phosphatidylserine (PS) at the inner leaflet is regulated by flippases 1 – 3 . When cells undergo apoptosis, PS translocates from the inner leaflet to the outer leaflet 4 . PS functions as a “eat me” signal, and macrophages engulf apoptotic cells displaying PS 5 . Moreover, the localization of lipids in the plasma membrane is regulated not only by proteins but also happens spontaneously. Cholesterol (Chol) is a steroid that is an essential component of the plasma membrane 6 , 7 . Chol plays an important role in forming a rigid domain in the plasma membrane, the so-called raft structure 8 . Fluid and rigid domains coexist in the plasma membrane. Fluid domains are composed of unsaturated phospholipids with cis double bonds, whereas rigid domains are composed of saturated phospholipids. Chol accumulates spontaneously into rigid domains and helps form raft structures. This domain plays an important role in regulating signal transduction through the plasma membrane; an example is the signal transduction mediated by epidermal growth factor receptor (EGFR) 9 . The mechanism of spontaneous Chol accumulation in membranes is unclear. S. L. Regen and co-workers studied the interactions between phospholipids and Chol using the nearest-neighbor recognition method using derivatives of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl- sn -glycero-3-phosphocholine (DPPC), and Chol. The nearest-neighbor interaction free energy between Chol derivative and POPC derivative is 0.0 ± 7.9 cal/mol in L o phase and + 160 ± 30 cal/mol in L d phase, and that between Chol derivative and DPPC derivative is − 260 ± 6.3 cal/mol in L o phase and + 12 ± 19 cal/mol in L d phase 10 . This difference suggests that Chol preferentially interacts with saturated phospholipids over unsaturated phospholipids with cis double bonds. Thus, unsaturated and saturated phospholipids push and pull Chol, facilitating lipid raft formation 11 . Although the formation of the plasma membrane is regulated by these mechanisms, other molecules can disrupt the plasma membrane. Bile acids are also steroid molecules, similar to Chol, but they typically disrupt the plasma membrane 12 , 13 . Figure 1 shows the chemical structures and schematic images of Chol and lithocholic acid (LCA), a bile acid. Bile acids can form micelles because of their carboxylic acid groups. The micelles suspend the dietary lipids and enhance their absorption. However, bile acids also suspend phospholipids in the plasma membrane, resulting in the disruption of the plasma membrane and cell death. Moreover, the geometrical structure of LCA differs from that of Chol because of the A/B ring junctions 14 . In unsaturated acyl chains of phospholipids, cis double bonds decrease lipid order 15 . Therefore, the bent structure of LCA may perturb the plasma membrane structure, resulting in cell death. However, the effects of the bent structure on plasma membranes have not been thoroughly investigated. In this study, the effects of the bent structure of LCA were investigated by comparing with those of Chol, which has a flat structure. To evaluate the effects of the bent structure and not of micellization, methylated LCA (Me-LCA) was employed (Fig. 1 ). To evaluate the localization of Me-LCA, pyrene-conjugated LCA (Py-LCA) and ( S )-(+)-4-( N , N -dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (DBD-APy)-conjugated LCA were used as fluorescent probes. Py-LCA was added to the thin lipid layers and giant unilamellar vesicles (GUVs) with the liquid crystalline phase and gel phase, and Me-LCA phase localization was evaluated. DBD-APy-LCA was added to the cells, and the cells were observed by confocal laser fluorescence microscopy. Liposomes were used to evaluate the effect of Me-LCA on the plasma membrane. The membrane fluidity and hydrophobicity of liposomes containing Me-LCA were evaluated, and the relationship between the effects on these properties and cytotoxicity was investigated. 2. Materials And Methods 2.1. Materials Phospholipids, 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC) and DPPC were purchased from NOF Corporation (Tokyo, Japan). Chol was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCA was purchased from the Tokyo Chemical Industry (Tokyo, Japan). To evaluate the localization of LCA derivatives, 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N -(lissamine rhodamine B sulfonyl) (ammonium salt) (Rho-DOPE), BODIPY conjugated Chol (BODIPY-Chol), Py-LCA, DRAQ5, Filipin III, and DBD-APy-LCA were employed as fluorescence probes. Rho-DOPE was purchased from Sigma-Aldrich (St. Louis, MO, USA). BODIPY-Chol and Filipin III were purchased from the Cayman Chemical Company (Ann Arbor, MI, USA). DRAQ5 was purchased from BioStatus, Ltd. (Loughborough, UK). Py-LCA and DBD-APy-LCA were synthesized by conjugation with 1-chloromethylpyrene and DBD-APy, respectively, purchased from Tokyo Chemical Industry (Tokyo, Japan). 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n -hydrate (DMT-MM) was conjugated with DBD-APy-LCA; it was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Fluorescence probes used to evaluate the membrane properties of liposomes, 1,6-diphenyl-1,3,5-hexatriene (DPH), and 6-dodecanoyl- N , N -dimethyl-2-naphthylamine (Laurdan) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The cytotoxicity of Chol and Me-LCA in phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 2 mM KH 2 PO 4 ; pH 7.3) was evaluated. Chol and Me-LCA were dissolved in PBS using MβCD, which was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Each solvent, N , N -dimethylformamide (DMF), diethyl ether (DEE), hydrochloric acid solution, n -hexane, methanol (MeOH), sulfuric acid (H 2 SO 4 ), hydrogen peroxide (H 2 O 2 ), chloroform, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and ethanol (EtOH) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), and 4 N HCl/dioxane was purchased from Watanabe Chemical Industries, Ltd. (Hiroshima, Japan). 2.2. Conjugation of pyrene with LCA Py-LCA was synthesized by conjugating LCA and 1-chloromethylpyrene ( Scheme S1 ). LCA (0.60 mmol), 1-chloromethylpyrene (0.60 mmol), and tetramethylammonium hydroxide pentahydrate (0.33 mmol) were added to DMF (3 mL). The mixture was then stirred for 50 min at 80°C. The products were extracted with DEE from diethyl ether-3.5-3.7% hydrochloric acid solution. DEE was removed using a rotary evaporator, and the products was added n -hexane (100 mL) and stirred overnight. The solution was filtered to remove the precipitate, and n -hexane was removed using a rotary evaporator. The product was dissolved in a small amount of MeOH (~ 3 mL) by ultrasonication. This solution was stored overnight at -20 ºC. The solution was filtered to obtain a precipitate and washed with cold EtOH. Purified Py-LCA was characterized by liquid-state 1 H NMR spectroscopy at 400 MHz (JNM-ECX-400 [JEOL Co. Ltd., Tokyo, Japan]) ( Fig. S1 ). 1 H-NMR (400 MHz, CDCl 3 ) δ 8.31–8.01 (m, 9H), 5.84 (s, 2H), 3.65–3.57 (m, 1H), 2.46–2.26 (m, 2H), 1.92 − 0.84 (m, 33H), 0.53 (s, 3H). 2.3. Synthesis of Me-LCA To methylate LCA, LCA (1 mmol) was dissolved in MeOH (4.5 mL), and 4 N HCl/dioxane (0.5 mL) was mixed ( Scheme S2 ). The solution was stirred overnight, followed by addition of excess water. The product was precipitated. The solvent was then filtered to collect the precipitate. The precipitate was dried under vacuum and characterized by liquid-state 1 H NMR at 400 MHz (JNM-ECX-400 [JEOL Co. Ltd., Tokyo, Japan]) ( Fig. S2 ). 1 H-NMR (400 MHz, CDCl 3 ) δ 3.66 (s, 3H), 3.66–3.58 (m, 1H), 2.39–2.17 (m, 2H), 1.98 − 0.86 (m, 33H), 0.63 (s, 3H). 2.4. Observation of lipid thin layer using fluorescence microscopy Thin lipid layers were prepared following a method described in a previous paper 16 . Glass coverslips (18 mm square) were cleaned with piranha solution (95 wt% H 2 SO 4 :30 wt% H 2 O 2 [4:1, v/v]) for 1 h at room temperature and washed extensively with water. The coverslips were stored in a desiccator and dried. Dried coverslips were placed in an electric muffle furnace TMF-5000 (Tokyo Rikakikai Co., Ltd., Tokyo, Japan). The temperature was increased from 25°C to 400°C for 1 h (6.3 ℃/min), and the coverslips were baked at 400°C for 3 h. DOPC, DPPC, Chol, and Me-LCA were dissolved in chloroform (5 µL). The concentration was set at 20 mg/mL. Fluorescence probe solutions of Rho-DOPE in chloroform (1 mM), BODIPY-Chol in DMSO (1 mM), and Py-LCA in THF (1 mM) were prepared. The fluorescence probes were mixed at a molar ratio of 1 mol%. The mixed solution was evaporated using a rotary evaporator, and the thin lipid layer was dried overnight in a desiccator. Water (150 µL) was added to the thin layer, and the suspension was subjected to five freeze-thaw cycles. The suspension was dropped on a coverslip and dried for 2 d in a humidified atmosphere at 60°C. The coverslip was dropped on 20 vol% MeOH, and a thin lipid layer was observed using fluorescence microscopy IX-51-11FL/PH-S (Olympus Corp., Tokyo, Japan). 2.5. Preparation and observation of GUVs The GUVs were prepared by gentle hydration using glucose 17 . DOPC, DPPC, Chol, and Me-LCA were dissolved in chloroform (50 µL) and the total lipid concentration was adjusted to 1 mM. Fluorescence probe solutions, Rho-DOPE in chloroform (1 mM), BODIPY-Chol in DMSO (1 mM), and Py-LCA in THF (1 mM), were added at a molar ratio of 1 mol%. Glucose dissolved in MeOH (20 mM, 25 µL) was added. The solution was added to a glass test tube and the solvent was removed using a rotary evaporator. The thin lipid layer was then dried in a desiccator. The thin lipid layer was then hydrated with water (500 µL) and incubated for 24 h at 60°C. The suspension was dropped on a depression slide and observed using fluorescence microscopy (IX-51-11FL/PH-S; Olympus Corp., Tokyo, Japan). 2.6. Cells and cell culture Hep G2 cells and HeLa cells (JCRB1054 and JCRB9004; JCRB Cell Bank, Osaka, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific; Waltham, MA, USA) in a humidified atmosphere of 5.0% CO 2 at 37°C. 2.7. Synthesis of DBD-APy-LCA The conjugation of ( S )-(+)-4-( N , N -dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (DBD-APy) with LCA was performed, as shown in Scheme S3 . DBD-APy (0.030 mmol), LCA (0.030 mmol), triethylamine (0.063 mmol), and diphenylphosphoryl azide (DPPA) (0.033 mmol) were dissolved in THF (1 mL). The solution was then stirred in the dark for 24 h. The organic solvent was removed using a rotary evaporator. The product was dried for 2–3 days in a desiccator. MeOH (3 mL) was then added to the product and kept for stirring overnight. The product was centrifuged, and MeOH (1 mL) was added to the product and centrifuged. The collected product was dried for 2–3 days in a desiccator. The synthesized DBD-APy-LCA was characterized by liquid-state 1 H NMR spectroscopy at 400 MHz (JNM-ECX-400 [JEOL Co. Ltd., Tokyo, Japan]) ( Fig. S3 ). 1 H-NMR (400 MHz, DMSO- d 6 ) δ 8.18 (d, J = 6.2 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 6.19 (d, J = 8.5 Hz, 1H), 4.46 (s, 1H), 4.41–4.36 (m, 1H), 2.66 (s, 6H), 2.28–2.20 (m, 2H), 2.13–0.80 (m, 33H), 0.47 (s, 3H). 2.8. Observation of cells treated with DBD-APy-LCA using a confocal fluorescence microscopy Hep G2 cells and HeLa cells (1.0×10 5 cells/mL) were cultured in 10% FBS containing DMEM (2 mL). After incubation for 24 h in a humidified atmosphere of 5% CO 2 at 37 ℃, the culture medium was replaced, and then 2 mL of 10% FBS containing DMEM and 5 µL of 7.6 mM Filipin III in DMSO was added, followed by incubation for 1 h. Then, 10 µL of 20 µM DBD-APy-LCA in DMSO and 2 µL of 5 mM DRAQ5 were added to the cells and incubated for 0.5 h. These cells were observed via confocal fluorescence laser microscopy (A1R+; Nikon, Tokyo, Japan) using the following filters: λ ex = 352 nm and λ em = 461 nm for Filipin III; λ ex = 504 nm and λ em = 511 nm for DBD-APy-LCA, and λ ex = 590–595 nm and λ em = 655–660 nm for DRAQ5. Moreover, the cells were cultured for 24 h, and 1.7 mM BODIPY-Chol in DMSO was added. These cells were observed after incubation for 0.5 h via confocal fluorescence laser microscopy using the following filters: λ ex = 504 nm and λ em = 511 nm for BODIPY-Chol. 2.9. Preparation of liposomes Model plasma membranes and liposomes were prepared using a previously described film hydration method 18 . The components of the liposomes (DOPC, DPPC, Chol, and Me-LCA) were dissolved in chloroform. The chloroform was removed using a rotary evaporator. After drying under vacuum overnight, the residual thin membrane was hydrated with water. The suspension was subjected to five freeze-thaw cycles and extruded to produce 100 nm diameter particles. 2.10. DPH fluorescence polarization measurement DPH was dissolved in THF, and its concentration was adjusted to 2.15 mM. The lipid concentration of the liposome suspensions was adjusted to 0.1 mM using water, and 1.85 µL of DPH solution was mixed with 10 mL liposome suspensions. The final concentrations of DPH and lipids was 0.4 µM and 0.1 mM (DPH:lipids = 1:250 [mol/mol]), and the volume ratio of THF in which DPH were dissolved was less than 1 vol%. Fluorescence intensity measurements and anisotropy evaluations were performed using an RF-5300PC fluorometer (Shimadzu Corp., Kyoto, Japan) equipped with polarizing plates. DPH was excited at 360 nm, and the fluorescence intensity at 430 nm was measured. When the fluorescence intensity was measured, the temperature was maintained at 10–50°C. The fluorescence polarization P value was calculated as follows: \(P=\frac{{I}_{vv}-\frac{{I}_{hv}}{{I}_{hh}}\times {I}_{vh}}{{I}_{vv}+\frac{{I}_{hv}}{{I}_{hh}}\times {I}_{vh}}\) (1) Where I is the fluorescence intensity 19 , the subscripts v and h indicate the orientation (vertical and horizontal) of the excitation and analyzer polarizers, respectively. 2.11. Evaluation of hydrophobicity using fluorescence probes The fluorescence spectra of some fluorescence probes are dependent on solvents, and this phenomenon is applied to evaluate the hydrophobicity of the vesicles. Laurdan is an environmentally responsive fluorescent probe, and its hydrophobicity was evaluated at the boundary surface between the hydrophilic and hydrophobic regions of vesicles (Fig. 2 ) 20 . Laurdan was dissolved in EtOH, and the concentration was adjusted to 1 mM. The lipid concentration of the liposome suspensions was adjusted to 0.1 mM using water, and 10 µL of Laurdan solution was mixed with 10 mL liposome suspensions. Final concentration of Laurdan and lipids was 1 µM and 0.1 mM (Laurdan:lipids = 1:100 [mol/mol]). The volume ratio of EtOH in which Laurdan was dissolved was less than 1 vol%. The fluorescence spectra of Laurdan were measured using an RF-5300PC fluorometer (Shimadzu Corp., Kyoto, Japan). Hydrophobicity was evaluated using the GP 340 values 21 – 23 . The values were calculated as follows: \({\text{G}\text{P}}_{340}=\frac{{I}_{440}-{I}_{490}}{{I}_{440}+{I}_{490}}\) (2) Where I 440 and I 490 are the fluorescence intensities at 440 and 490 nm, respectively, when Laurdan was excited at 340 nm. Pyrene is also an environmentally responsive fluorescent probe 24 , 25 , and pyrene is used to evaluate the hydrophobicity of micelles 26 . Moreover, conjugation of dicarboxylic acids (succinic acid, suberic acid, and dodecanedioic acid) can evaluate gradual hydrophobicity in the hydrophobic region (Fig. 2 ) 27 . The pyrene-succinic acid conjugate (Py-C 3 -COOH), pyrene-suberic acid conjugate (Py-C 7 -COOH), and pyrene-dodecanedioic acid conjugate (Py-C 11 -COOH) were dissolved in THF, and their concentrations were adjusted to 10 mM. The solution was then mixed with chloroform to dissolve the lipids. Liposomes were prepared and adjusted to 100 nm as described above. The ratio of pyrene-dicarboxylic acid conjugates to lipids was 1:100 [mol/mol]. The prepared liposome suspension was diluted, and the final concentrations of pyrene − dicarboxylic acid conjugate and lipids were 1 µM and 0.1 mM. Pyrene-dicarboxylic acid conjugates were excited at 336 nm. Fluorescence spectra were measured from 350 to 600 nm at 10–50°C using an RF-5300PC fluorometer (Shimadzu Corp., Kyoto, Japan). Hydrophobicity was evaluated based on the fluorescence intensity of peaks I (377 ± 2 nm, I I ) and III (387 ± 2 nm, I III ). The ratio of I I to I III ( I I / I III ) is related to the relative permittivity of aliphatic monoalcohols, and the relative permittivity of the vesicles can be estimated from the I I / I III values based on correlation equations. 2.12. MTT assay The MTT assay was performed using the MTT Cell Count Kit (Nacalai Tesque, Kyoto, Japan). Hep G2 cells and HeLa cells were seeded on 96-well culture plates (100 µL, 2.0×10 5 cells/mL) and cultured in DMEM supplemented with 10% FBS in an incubator. After 24 h of growth, the following formulations (3 µL) were added to the cells: Chol and Me-LCA diluted in PBS and treated with 200 mM MβCD (Sigma-Aldrich [St. Louis, MO, USA]). After incubation for 24 h, 10 µL MTT solution was added to the cells. After incubation for 3 h, 100 µL of solubilization solution was added and the formazan precipitate was dissolved by pipetting. The absorbance of each well at 570 and 650 nm was measured using a microplate spectrophotometer (Flex Station [Molecular Devices; San Jose, CA, USA]). Cell viability was calculated using the following equation: \(\text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e} \text{c}\text{e}\text{l}\text{l} \text{v}\text{i}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}=\frac{{A}_{570}-{A}_{650}}{{{A}_{570}}_{\left(0\right)}-{{A}_{650}}_{\left(0\right)}}\) (3) Where A 570 and A 650 are the absorbance values at 570 and 650 nm, respectively, when the cells were treated with the sample. A 570(0) and A 650(0) are the absorbance values at 570 and 650 nm, respectively, when the cells were treated with 200 mM MβCD in PBS. 3. Results 3.1. Localization of Me-LCA in phospholipid layers and plasma membrane Fluorescence probes, Rho-DOPE, BODIPY-Chol, and Py-LCA were mixed in thin lipid layers, and the layers were observed by fluorescence microscopy (Fig. 3 (a) ). When a thin lipid layer composed of DOPC/DPPC/Chol (1:1:1, mol/mol) was observed, the DOPC-rich L d phase and DPPC- and Chol-rich L o phases coexisted on the liposomal membrane at ~ 25 ℃ 28 – 30 . Rho-DOPE was localized in the L d phase, whereas BODIPY-Chol was localized in the L o phase. The fluorescence of Rho-DOPE and BODIPY-Chol was observed on different parts of the thin lipid layer because of phase separation. Py-LCA fluorescence was observed in the L d phase. This result suggests that Me-LCA is localized in the L d phase, even though Me-LCA is a steroid molecule similar to Chol, which is localized in the L o phase. After excessive secretion of bile acids, they may accumulate in the plasma membrane. To investigate the behavior of Me-LCA in plasma membranes containing a large number of Me-LCA molecules, DOPC/DPPC/Chol/Me-LCA (1:1:1:1, mol/mol) lipid thin layers were also employed. The fluorescence of Rho-DOPE and Py-LCA was observed in the same part of the lipid thin layer. These fluorescence probes and BODIPY-Chol were observed in different parts of the lipid thin layer. Thus, the thin lipid layer was heterogeneous despite the presence of a large number of Me-LCA molecules (25 mol%), and Py-LCA was localized in the L d phase. Moreover, Py-LCA localization was evaluated using GUVs (Fig. 3 (b) ). This result using GUVs was similar to that obtained using thin lipid layers. Py-LCA was localized in the L d phase of GUVs composed of DOPC, DPPC, and Chol. Me-LCA did not affect phase separation, and Py-LCA was localized in the L d phase of GUVs composed of DOPC, DPPC, Chol, and Me-LCA. DBD-APy-LCA was also used to investigate the cellular localization of Me-LCA. DBD-APy-LCA was added to Hep G2 cells and HeLa cells, and these cells were observed by confocal fluorescence microscopy. These cells were also treated with DRAQ5 as a fluorescent DNA stain and Filipin III as a fluorescent Cholstain. Figure 4 shows confocal fluorescence microscopic images of Hep G2 cells and HeLa cells that were treated with these fluorescence probes. The fluorescence of DBD-APy-LCA was observed in the plasma membrane, similar to that of Filipin III. Moreover, DBD-APy-LCA was also observed in the cytoplasm. This result shows that some DBD-APy-LCA molecules are localized in the plasma membrane, whereas other molecules are localized in the cytoplasm. This result suggests that Me-LCA accumulates in the plasma membrane and the organelle membranes. 3.2. Evaluation of effects of Me-LCA on membrane properties and comparison with Chol DPH fluorescence polarization measurements were employed to evaluate the microviscosity of liposomes 19 . Figure 5 (a) shows the fluorescence polarization P values of DOPC liposomes containing Chol and Me-LCA. The P values of the DOPC liposomes increased with increasing Chol molar ratio at 10°C. An increase in temperature reduced the P values of DOPC liposomes regardless of the Chol molar ratio, but the P values of DOPC liposomes increased with increasing Chol molar ratio, independent of temperature. On the other hand, there were hardly any differences in the P values regardless of the Me-LCA molar ratio at 10–50°C. Figure 5 (b) shows P values of DPPC liposomes. The P values of DPPC liposomes were similar regardless of whether steroid molecules containing or not at ≤ 37 ℃. The P values were different at 50°C for the DPPC liposomes. Although the P value of DPPC liposomes without Chol remarkably decreased with a change in temperature from 37°C to 50°C, this decrease was suppressed by Chol. The smallest decrease was observed for the 50 mol% Chol. The P values of DPPC liposomes with 0–50 mol% Me-LCA were also measured, but no difference was observed regardless of the Me-LCA molar ratio, unlike Chol at 10–50°C. The P value of DPPC liposomes with 0 mol% Me-LCA remarkably decreased with a change in temperature from 37°C to 50°C, and the P values of DPPC liposomes with 10–50 mol% Me-LCA similarly changed. Figure 5 (c) shows the P values of DOPC/DPPC/Chol (1:1:1, mol/mol) liposomes with 0–25 mol% Me-LCA. The P values were similar regardless of the Me-LCA molar ratio and temperature. The P values decreased with increasing temperature, similar to the DOPC liposomes with Me-LCA. Hydrophobicity at the boundary surface between the headgroup and acyl chain region was evaluated using a Laurdan fluorescence probe. Laurdan was used to evaluate hydrophobicity based on GP 340 values calculated from the fluorescence spectrum 21 – 23 . Figure 6 (a) shows the GP 340 values of DOPC liposomes with Chol or Me-LCA. Chol increased GP 340 values, whereas Me-LCA did not affect GP 340 values in a temperature-dependent manner. Figure 6 (b) shows GP 340 values of DPPC liposomes. Differences of GP 340 values did not be observed regardless of whether steroid molecules containing or not at < 40 ℃. However, Chol increased the GP 340 values at 50°C, whereas Me-LCA did not affect GP 340 values. Figure 6 (c) shows the GP 340 values of DOPC/DPPC/Chol (1:1:1, mol/mol) liposomes; Me-LCA did not affect GP 340 values at 10–50°C. Hydrophobicity in the hydrophobic region was also evaluated using fluorescence probes and pyrene-dicarboxylic acid conjugates (Py-C 3 -COOH, Py-C 7 -COOH, and Py-C 11 -COOH). Pyrene was used to evaluate hydrophobicity based on I I / I III values, which are the ratio of the intensities of peak I (377 ± 2 nm, I I ) and peak III (387 ± 2 nm, I III ) of the pyrene fluorescence spectrum. The I I / I III values were used to estimate the relative permittivity, and Py-C 3 -COOH, Py-C 7 -COOH, and Py-C 11 -COOH were evaluated in the shallow, middle, and deep regions 27 . Figure 7 shows the estimated ε r of the liposomes using Py-C 7 -COOH. The estimated ε r of DOPC liposomes with 0–50 mol% Chol was 8–16 at 10–50°C, and a remarkable difference was hardly observed, regardless of Chol. In addition, Me-LCA did not affect the estimated ε r of the DOPC liposomes. Moreover, the estimated ε r values of DPPC liposomes and DOPC/DPPC/Chol (1:1:1, mol/mol) liposomes were similar regardless of the molar ratio of Chol and Me-LCA. Fig. S5 and Fig. S6 show the estimated ε r values of liposomes using Py-C 3 -COOH and Py-C 11 -COOH. These results also show that Me-LCA did not significantly affect the estimated ε r of the liposomes. 3.3. Cytotoxicity of Me-LCA Hep G2 cells and HeLa cells were treated with Chol or Me-LCA, and the cell viability of these cells was evaluated using the MTT assay (Fig. 8 ). When Hep G2 cells were treated with Chol and Me-LCA at concentrations < 100 µM, Chol and Me-LCA did not affect relative cell viability. High concentrations of Chol and Me-LCA decreased the relative cell viability. In contrast, the relative viability of HeLa cells showed different changes after treatment with Chol or Me-LCA. When HeLa cells were treated with Chol at concentrations < 150 µM, relative cell viability increased. The relative cell viability was decreased from 2.4 to 1.2 at high concentrations. However, Me-LCA did not increase relative cell viability. Changes in the relative viability of HeLa cells treated with Me-LCA were similar to those of Hep G2 cells. Treatment with 300 µM Me-LCA did not affect the relative viability of HeLa cells, and a high concentration of Me-LCA decreased the relative cell viability. 4. Discussion The cytotoxicity of bile acids is well known. A detergent model of cell death induced by bile acids posits that bile acids remove phospholipids from the plasma membrane 12 , 13 . In this study, we investigated the possibility that the bent structure of bile acids contributes to cell death. Me-LCA, which cannot form micelles, was used as a model bile acid. The localization of Me-LCA was initially investigated because the effects of Me-LCA on the lipid bilayer may depend on the phase state. To evaluate the effects of Me-LCA, Chol, which has a flat structure, was used. It has been referred about a relationship between localization of Chol and effects of Chol on the lipid bilayer. The condensation effect of Chol can produce many close hydrophobic contacts with the neighboring acyl chains of phospholipids, resulting in tight packing 31 , 32 . Moreover, Chol becomes a repulsive phospholipid as the proportion of cis double bonds increases 33 . Thus, Chol pulls saturated phospholipids and pushes unsaturated phospholipids with cis double bonds away, inducing phase separation 11 . Other steroid molecules such as 7-dehydrocholesterol, campesterol, β-sitosterol, ergosterol, brassicasterol, and stigmasterol also increase the lipid chain order 34 . The condensation effect was not unique to Chol. In contrast, Me-LCA, a steroid molecule derivative, did not increase the lipid chain order. As shown in Fig. 5 , the membrane fluidity was evaluated using DPH. Me-LCA did not affect the P value, whereas Chol decreased the P value. The difference between these steroid molecules containing Chol and Me-LCA is their geometrical structure. We assume that the flat structure of Chol is suitable for interaction with the neighboring acyl chains of phospholipids, but the bent structure of Me-LCA is less close to the hydrophobic contacts. This difference was also related to localization in the lipid bilayer. BODIPY-Chol localized in the L o phase, while Py-LCA localized in the L d phase (Fig. 3 ). This result suggests that Chol prefers saturated phospholipids to bile acids, and that Chol pushes bile acids to the L d phase. When cells were treated with DBD-APy-LCA, fluorescence was observed in both the plasma membrane and the cytosol (Fig. 4 ). DBD-APy-LCA is taken up into the cytosol by endocytosis and/or bile acid receptors. Filipin III binds to Chol in the plasma membrane, resulting in its fluorescence in the plasma membrane. However, when the cells were treated with BODIPY-Chol, fluorescence was observed in the cytosol ( Fig. S4 ). Chol is delivered by apolipoproteins in the body, and its distribution is regulated 35 . In contrast, the distribution of dissolved Chol molecules in DMSO was not regulated compared with Chol in the body. Dissolved Chol molecules are distributed in the cytosol via the plasma membrane. Therefore, both dissolved steroid molecules affect the properties of the plasma membrane and those of the organelle membranes. Although Me-LCA did not condense phospholipids, it did not perturb the lipid bilayer. When the lipid bilayer is perturbed by other molecules, such as amphiphilic peptides, the P value decrease 36 . This perturbation promotes the penetration of water molecules into the lipid bilayer 37 . Thus, it suggested that the GP 340 value and the estimated ε r decreased. However, Me-LCA did not affect P value, GP 340 value, or estimated ε r (Fig. 5 , Fig. 6 , and Fig. 7 ). These results suggested that Me-LCA with a bent structure hardly perturbed the lipid bilayer. These results are related to cytotoxicity. Me-LCA did not affect proliferation of Hep G2 cells and HeLa cells. Deoxycholic acid (DC), chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA) at 750 µM can lyse Hep G2 cells after 4 h of incubation 38 . As shown in Fig. 8 , the relative cell viability of Hep G2 cells and HeLa cells treated with Me-LCA at > 750 µM was higher than that of Hep G2 cells treated with DC, CDCA, and GCDCA at 750 µM. Evaluation of cytotoxicity suggested that by removing phospholipids constituting the plasma membrane, bile acid micelles mainly contribute to cell death. Notably, Me-LCA cannot replace Chol. Chol is a component of raft structure, which plays an important role in regulating the behavior of membrane proteins 8 . When HeLa cells were treated with Chol, their proliferation improved (Fig. 8 ). This phenomenon has been reported in human prostate cancer cells (PC-3 cells) 39 . EGFR is a membrane protein that promotes cellular proliferation. The EGFR substrate 15-related protein (EPS15R) regulates the internalization of EGFR 40 , resulting in the inhibition of proliferation. Chol affects EGFR internalization. Adipocyte plasma membrane-associated protein (APMAP) is a transmembrane protein that accumulates in Chol-induced lipid rafts. APMAP interacts with EPS15R. Interaction with EPS15R inhibited the internalization of EGFR and promoted proliferation. 5. Conclusions A methylated bile acid derivative, Me-LCA, was used to investigate the cytotoxicity induced by bile acids. Me-LCA localized in the L d phase and did not perturb the lipid bilayer. Me-LCA also did not affect cell proliferation. These results show that the removal of phospholipids from the plasma membrane is mainly responsible for the cytotoxicity induced by bile acids. Moreover, the effect of Me-LCA was compared with that of Chol, a steroid molecule similar to bile acids. Chol ordered the lipid array. Me-LCA did not perturb the membrane and order it. Chol increased the proliferation of HeLa cells, whereas Me-LCA did not. These results suggest that Me-LCA is not an alternative to Chol that shows a condensation effect and is a component of the raft structure. Me-LCA did not affect membrane properties to induce cell death. Declarations Acknowledgement This study was supported by the Division of Medical Research Support of the Advanced Research Support Center at Ehime University. We would like to thank Editage (www.editage.com) for English language editing. Author contributions T.I. wrote the main manuscript text and prepared figures 4 and 8. N.E. prepared figures 3, 5 and 7. Y.N. prepared a figure 6. T.K. synthesized and characterized Me-LCA and fluorescence probes. K.H. and H.N. are supervisors and edited the manuscript. All authors read and approved the final manuscript. Data availability statement The data generated during the study are available from the corresponding author, Keita Hayashi, upon reasonable request. Competing Interests Statement The authors declare no competing interests. References Tanaka, K., Fujimura-Kamada, K. & Yamamoto, T. Functions of phospholipid flippases. J. Biochem. 149 , 131-143 (2010). Tang, X., Halleck, M. S., Schlegel, R. A. & Williamson, P. A Subfamily of P-Type ATPases with Aminophospholipid Transporting Activity. Science 272 , 1495-1497 (1996). Yabas, M. et al. ATP11C is critical for the internalization of phosphatidylserine and differentiation of B lymphocytes. Nat. Immunol. 12 , 441-449 (2011). Leventis, P. A. & Grinstein, S. The Distribution and Function of Phosphatidylserine in Cellular Membranes. Annu. Rev. Biophys. 39 , 407-427 (2010). Nagata, S., Hanayama, R. & Kawane, K. Autoimmunity and the Clearance of Dead Cells. Cell 140 , 619-630 (2010). Harayama, T. & Riezman, H. Understanding the diversity of membrane lipid composition. Nat. Rev. Mol. Cell Biol. 19 , 281-296 (2018). Van Meer, G., Voelker, D. R. & Feigenson, G. W. Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9 , 112-124 (2008). Sezgin, E., Levental, I., Mayor, S. & Eggeling, C. The mystery of membrane organization: composition, regulation and roles of lipid rafts. Nat. Rev. Mol. Cell Biol. 18 , 361-374 (2017). Simons, K. & Toomre, D. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1 , 31-39 (2000). Wang, C., Krause, M. R. & Regen, S. L. Push and Pull Forces in Lipid Raft Formation: The Push Can Be as Important as the Pull. J. Am. Chem. Soc. 137 , 664-666 (2015). Krause, M. R. & Regen, S. L. The Structural Role of Cholesterol in Cell Membranes: From Condensed Bilayers to Lipid Rafts. Acc. Chem. Res. 47 , 3512-3521 (2014). Hofmann, A. F. Bile acids: the good, the bad, and the ugly. News Physiol. Sci. 14 , 24-29 (1999). Hofmann, A. F. & Hagey, L. Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics. Cell. Mol. Life Sci. 65 , 2461-2483 (2008). Reschly, E. J. et al. Evolution of the bile salt nuclear receptor FXR in vertebrates. J. Lipid Res. 49 , 1577-1587 (2008). Ichimori, H., Hata, T., Matsuki, H. & Kaneshina, S. Effect of unsaturated acyl chains on the thermotropic and barotropic phase transitions of phospholipid bilayer membranes. Chem. Phys. Lipids 100 , 151-164 (1999). Tayebi, L. et al. Long-range interlayer alignment of intralayer domains in stacked lipid bilayers. Nat. Mater. 11 , 1074-1080 (2012). Tsumoto, K., Matsuo, H., Tomita, M. & Yoshimura, T. Efficient formation of giant liposomes through the gentle hydration of phosphatidylcholine films doped with sugar. Colloids Surf. B 68 , 98-105 (2009). Hayashi, K. et al. Effect of dehydrocholic acid conjugated with a hydrocarbon on a lipid bilayer composed of 1,2-dioleoyl- sn -glycero-3-phosphocholine. Colloids Surf. B 181 , 58-65 (2019). Borenstain, V. & Barenholz, Y. Characterization of liposomes and other lipid assemblies by multiprobe fluorescence polarization. Chem. Phys. Lipids 64 , 117-127 (1993). Osella, S., Smisdom, N., Ameloot, M. & Knippenberg, S. Conformational changes as driving force for phase recognition: The case of Laurdan. Langmuir 35 , 11471-11481 (2019). Parasassi, T. & Gratton, E. Membrane lipid domains and dynamics as detected by Laurdan fluorescence. J. Fluoresc. 5 , 59-69 (1995). Parasassi, T., De Stasio, G., d'Ubaldo, A. & Gratton, E. Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence. Biophys. J. 57 , 1179-1186 (1990). Parasassi, T., De Stasio, G., Ravagnan, G., Rusch, R. & Gratton, E. Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence. Biophys. J. 60 , 179-189 (1991). Dong, D. C. & Winnik, M. A. The Py scale of solvent polarities. Solvent effects on the vibronic fine structure of pyrene fluorescence and empirical correlations with ET and Y values. Photochem. Photobiol. 35 , 17-21 (1982). Dong, D. C. & Winnik, M. A. The Py scale of solvent polarities. Can. J. Chem. 62 , 2560-2565 (1984). Kalyanasundaram, K. & Thomas, J. Environmental effects on vibronic band intensities in pyrene monomer fluorescence and their application in studies of micellar systems. J. Am. Chem. Soc. 99 , 2039-2044 (1977). Hayashi, K. et al. Quantitative Determination of Relative Permittivity Based on the Fluorescence Property of Pyrene Derivatives: An Interpretation of Hydrophobicity in Self-Assembled Aggregates of Nonionic Amphiphiles. J. Phys. Chem. B 125 , 6192-6200 (2021). Marsh, D. Cholesterol-induced fluid membrane domains: a compendium of lipid-raft ternary phase diagrams. Biochim. Biophys. Acta Biomembr. 1788 , 2114-2123 (2009). Davis, J. H., Clair, J. J. & Juhasz, J. Phase equilibria in DOPC/DPPC-d 62 /cholesterol mixtures. Biophys. J. 96 , 521-539 (2009). Veatch, S. L. & Keller, S. L. Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol. Biophys. J. 85 , 3074-3083 (2003). Meyer, F. d. & Smit, B. Effect of cholesterol on the structure of a phospholipid bilayer. Proc. Natl. Acad. Sci. USA. 106 , 3654-3658 (2009). Róg, T., Pasenkiewicz-Gierula, M., Vattulainen, I. & Karttunen, M. Ordering effects of cholesterol and its analogues. Biochim. Biophys. Acta Biomembr. 1788 , 97-121 (2009). Almeida, P. F. How to Determine Lipid Interactions in Membranes from Experiment Through the Ising Model. Langmuir 35 , 21-40 (2019). Shaghaghi, M., Chen, M.-T., Hsueh, Y.-W., Zuckermann, M. J. & Thewalt, J. L. Effect of Sterol Structure on the Physical Properties of 1-Palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine Membranes Determined Using 2H Nuclear Magnetic Resonance. Langmuir 32 , 7654-7663 (2016). Feng, J. et al. Caffeine-free hawk tea lowers cholesterol by reducing free cholesterol uptake and the production of very-low-density lipoprotein. Commun. Biol. 2 , 173 (2019). Raghuraman, H. & Chattopadhyay, A. Interaction of Melittin with Membrane Cholesterol: A Fluorescence Approach. Biophys. J. 87 , 2419-2432 (2004). Vijayan, K., Discher, D. E., Lal, J., Janmey, P. & Goulian, M. Interactions of Membrane-Active Peptides with Thick, Neutral, Nonzwitterionic Bilayers. J. Phys. Chem. B 109 , 14356-14364 (2005). Pérez-Pineda, S. I., Baylón-Pacheco, L., Espíritu-Gordillo, P., Tsutsumi, V. & Rosales-Encina, J. L. Effect of bile acids on the expression of MRP3 and MRP4: An In vitro study in HepG2 cell line. Ann. Hepatol. 24 , 100325 (2021). Jiang, S. et al. Cholesterol Induces Epithelial-to-Mesenchymal Transition of Prostate Cancer Cells by Suppressing Degradation of EGFR through APMAP. Cancer Res. 79 , 3063-3075 (2019). Carbone, R. et al. eps15 and eps15R Are Essential Components of the Endocytic Pathway. Cancer Res. 57 , 5498-5504 (1997). Additional Declarations No competing interests reported. Supplementary Files 220602FigS1.3.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1700469","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":112575671,"identity":"7caa6708-fbe2-4338-bda2-3f1dc28eabf5","order_by":0,"name":"Tomoyuki Iwasaki","email":"","orcid":"","institution":"Ehime University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tomoyuki","middleName":"","lastName":"Iwasaki","suffix":""},{"id":112575672,"identity":"eb7ad14b-009a-41c8-8f82-d38b6720a747","order_by":1,"name":"Nobuyuki Endo","email":"","orcid":"","institution":"National Institute of Technology, Nara 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Me-LCA.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1700469/v1/149d319c322d74b81994f14f.png"},{"id":22791570,"identity":"b1a3e2fe-86eb-4664-8090-9365f8ad4807","added_by":"auto","created_at":"2022-06-17 19:57:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":131396,"visible":true,"origin":"","legend":"\u003cp\u003eLocalization of fluorescence probes in liposome.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-1700469/v1/453a99aed4014e94849d0e8f.png"},{"id":22791779,"identity":"ed20a2bb-6f42-444a-9a3d-63ae662e4f17","added_by":"auto","created_at":"2022-06-17 20:02:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1805001,"visible":true,"origin":"","legend":"\u003cp\u003eObservation of Rho-DOPE (red), BODIPY-Chol (green), and Py-LCA (blue) in (a) lipid thin layers and (b) GUVs using a fluorescence microscopy.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1700469/v1/acb14ae6cbb120551c42b4d4.png"},{"id":22791778,"identity":"6e148f80-7e5c-4bdb-89b4-3bf69a5bc411","added_by":"auto","created_at":"2022-06-17 20:02:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1664541,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal fluorescence microscopy images of (a) Hep G2 cells and (b) HeLa cells treated with DRAQ5 (red), Filipin III (blue), and DBD-APy-LCA (green).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-1700469/v1/981e0c182677087f2c59c68a.png"},{"id":22791575,"identity":"517c7ea6-6183-4abb-b26a-629574002844","added_by":"auto","created_at":"2022-06-17 19:57:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":199092,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eP\u003c/em\u003e values of (a) DOPC liposome, (b) DPPC liposome, and (c) DOPC/DPPC/Chol (1:1:1, mol/mol) liposome with Me-LCA.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-1700469/v1/4ce00cb8e2c6fd469e28aca4.png"},{"id":22791578,"identity":"c08730af-6f1a-4c8f-8b1b-24bc0b97554e","added_by":"auto","created_at":"2022-06-17 19:57:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":204141,"visible":true,"origin":"","legend":"\u003cp\u003eGP\u003csub\u003e340\u003c/sub\u003e values of (a) DOPC liposome, (b) DPPC liposome, and (c) DOPC/DPPC/Chol (1:1:1, mol/mol) liposome with Me-LCA.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-1700469/v1/b3e275eae0c33d1bfdc784cb.png"},{"id":22791777,"identity":"7cb316d9-0191-4e3d-b1c9-601f27405153","added_by":"auto","created_at":"2022-06-17 20:02:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":205319,"visible":true,"origin":"","legend":"\u003cp\u003eEstimated \u003cem\u003eε\u003c/em\u003e\u003csub\u003er\u003c/sub\u003e values using Py-C\u003csub\u003e7\u003c/sub\u003e-COOH of (a) DOPC liposome, (b) DPPC liposome, and (c) DOPC/DPPC/Chol (1:1:1, mol/mol) liposome with Me-LCA.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-1700469/v1/d03e5360a39ff6668e355b5d.png"},{"id":22791573,"identity":"cd9e6c65-3fb8-4956-8792-755738b5fad2","added_by":"auto","created_at":"2022-06-17 19:57:21","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":65540,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of (a) Hep G2 cells and (b) HeLa cells treated with Chol or Me-LCA.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-1700469/v1/7cd4503a36ffcf1141c4d464.png"},{"id":25917098,"identity":"3aa2aef1-4f79-4b2f-bddd-450ed54cf977","added_by":"auto","created_at":"2022-09-01 06:44:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2301288,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1700469/v1/c1067af1-cba2-4b31-9cfa-caf35f2a90b1.pdf"},{"id":22791576,"identity":"fc52f6cb-6b9d-4fb6-8989-c8369e87ab61","added_by":"auto","created_at":"2022-06-17 19:57:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6662792,"visible":true,"origin":"","legend":"","description":"","filename":"220602FigS1.3.docx","url":"https://assets-eu.researchsquare.com/files/rs-1700469/v1/973779bfaf2dbdc05926aba7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Effect of Methylated Lithocholic Acid on Plasma Membrane Properties: The Difference with the Effect of Cholesterol","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe plasma membrane is composed of several types of lipids. The localization of these lipids in the plasma membrane is regulated, and these lipids play key biological roles. For example, the localization of phosphatidylserine (PS) at the inner leaflet is regulated by flippases\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. When cells undergo apoptosis, PS translocates from the inner leaflet to the outer leaflet\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. PS functions as a \u0026ldquo;eat me\u0026rdquo; signal, and macrophages engulf apoptotic cells displaying PS\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Moreover, the localization of lipids in the plasma membrane is regulated not only by proteins but also happens spontaneously. Cholesterol (Chol) is a steroid that is an essential component of the plasma membrane\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Chol plays an important role in forming a rigid domain in the plasma membrane, the so-called raft structure\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Fluid and rigid domains coexist in the plasma membrane. Fluid domains are composed of unsaturated phospholipids with \u003cem\u003ecis\u003c/em\u003e double bonds, whereas rigid domains are composed of saturated phospholipids. Chol accumulates spontaneously into rigid domains and helps form raft structures. This domain plays an important role in regulating signal transduction through the plasma membrane; an example is the signal transduction mediated by epidermal growth factor receptor (EGFR)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe mechanism of spontaneous Chol accumulation in membranes is unclear. S. L. Regen and co-workers studied the interactions between phospholipids and Chol using the nearest-neighbor recognition method using derivatives of 1-palmitoyl-2-oleoyl-\u003cem\u003esn\u003c/em\u003e-glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-\u003cem\u003esn\u003c/em\u003e-glycero-3-phosphocholine (DPPC), and Chol. The nearest-neighbor interaction free energy between Chol derivative and POPC derivative is 0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.9 cal/mol in L\u003csub\u003eo\u003c/sub\u003e phase and +\u0026thinsp;160\u0026thinsp;\u0026plusmn;\u0026thinsp;30 cal/mol in L\u003csub\u003ed\u003c/sub\u003e phase, and that between Chol derivative and DPPC derivative is \u0026minus;\u0026thinsp;260\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3 cal/mol in L\u003csub\u003eo\u003c/sub\u003e phase and +\u0026thinsp;12\u0026thinsp;\u0026plusmn;\u0026thinsp;19 cal/mol in L\u003csub\u003ed\u003c/sub\u003e phase\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This difference suggests that Chol preferentially interacts with saturated phospholipids over unsaturated phospholipids with \u003cem\u003ecis\u003c/em\u003e double bonds. Thus, unsaturated and saturated phospholipids push and pull Chol, facilitating lipid raft formation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough the formation of the plasma membrane is regulated by these mechanisms, other molecules can disrupt the plasma membrane. Bile acids are also steroid molecules, similar to Chol, but they typically disrupt the plasma membrane\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the chemical structures and schematic images of Chol and lithocholic acid (LCA), a bile acid. Bile acids can form micelles because of their carboxylic acid groups. The micelles suspend the dietary lipids and enhance their absorption. However, bile acids also suspend phospholipids in the plasma membrane, resulting in the disruption of the plasma membrane and cell death. Moreover, the geometrical structure of LCA differs from that of Chol because of the A/B ring junctions\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In unsaturated acyl chains of phospholipids, \u003cem\u003ecis\u003c/em\u003e double bonds decrease lipid order\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Therefore, the bent structure of LCA may perturb the plasma membrane structure, resulting in cell death. However, the effects of the bent structure on plasma membranes have not been thoroughly investigated.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this study, the effects of the bent structure of LCA were investigated by comparing with those of Chol, which has a flat structure. To evaluate the effects of the bent structure and not of micellization, methylated LCA (Me-LCA) was employed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To evaluate the localization of Me-LCA, pyrene-conjugated LCA (Py-LCA) and (\u003cem\u003eS\u003c/em\u003e)-(+)-4-(\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (DBD-APy)-conjugated LCA were used as fluorescent probes. Py-LCA was added to the thin lipid layers and giant unilamellar vesicles (GUVs) with the liquid crystalline phase and gel phase, and Me-LCA phase localization was evaluated. DBD-APy-LCA was added to the cells, and the cells were observed by confocal laser fluorescence microscopy. Liposomes were used to evaluate the effect of Me-LCA on the plasma membrane. The membrane fluidity and hydrophobicity of liposomes containing Me-LCA were evaluated, and the relationship between the effects on these properties and cytotoxicity was investigated.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1. Materials\u003c/h2\u003e\n \u003cp\u003ePhospholipids, 1,2-dioleoyl-\u003cem\u003esn\u003c/em\u003e-glycero-3-phosphocholine (DOPC) and DPPC were purchased from NOF Corporation (Tokyo, Japan). Chol was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCA was purchased from the Tokyo Chemical Industry (Tokyo, Japan). To evaluate the localization of LCA derivatives, 1,2-dioleoyl-\u003cem\u003esn\u003c/em\u003e-glycero-3-phosphoethanolamine-\u003cem\u003eN\u003c/em\u003e-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rho-DOPE), BODIPY conjugated Chol (BODIPY-Chol), Py-LCA, DRAQ5, Filipin III, and DBD-APy-LCA were employed as fluorescence probes. Rho-DOPE was purchased from Sigma-Aldrich (St. Louis, MO, USA). BODIPY-Chol and Filipin III were purchased from the Cayman Chemical Company (Ann Arbor, MI, USA). DRAQ5 was purchased from BioStatus, Ltd. (Loughborough, UK). Py-LCA and DBD-APy-LCA were synthesized by conjugation with 1-chloromethylpyrene and DBD-APy, respectively, purchased from Tokyo Chemical Industry (Tokyo, Japan). 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride \u003cem\u003en\u003c/em\u003e-hydrate (DMT-MM) was conjugated with DBD-APy-LCA; it was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Fluorescence probes used to evaluate the membrane properties of liposomes, 1,6-diphenyl-1,3,5-hexatriene (DPH), and 6-dodecanoyl-\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-dimethyl-2-naphthylamine (Laurdan) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The cytotoxicity of Chol and Me-LCA in phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, and 2 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e; pH 7.3) was evaluated. Chol and Me-LCA were dissolved in PBS using M\u0026beta;CD, which was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Each solvent, \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-dimethylformamide (DMF), diethyl ether (DEE), hydrochloric acid solution, \u003cem\u003en\u003c/em\u003e-hexane, methanol (MeOH), sulfuric acid (H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), chloroform, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and ethanol (EtOH) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), and 4 N HCl/dioxane was purchased from Watanabe Chemical Industries, Ltd. (Hiroshima, Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2. Conjugation of pyrene with LCA\u003c/h2\u003e\n \u003cp\u003ePy-LCA was synthesized by conjugating LCA and 1-chloromethylpyrene (\u003cstrong\u003eScheme S1\u003c/strong\u003e). LCA (0.60 mmol), 1-chloromethylpyrene (0.60 mmol), and tetramethylammonium hydroxide pentahydrate (0.33 mmol) were added to DMF (3 mL). The mixture was then stirred for 50 min at 80\u0026deg;C. The products were extracted with DEE from diethyl ether-3.5-3.7% hydrochloric acid solution. DEE was removed using a rotary evaporator, and the products was added \u003cem\u003en\u003c/em\u003e-hexane (100 mL) and stirred overnight. The solution was filtered to remove the precipitate, and \u003cem\u003en\u003c/em\u003e-hexane was removed using a rotary evaporator. The product was dissolved in a small amount of MeOH (~\u0026thinsp;3 mL) by ultrasonication. This solution was stored overnight at -20 \u0026ordm;C. The solution was filtered to obtain a precipitate and washed with cold EtOH. Purified Py-LCA was characterized by liquid-state \u003csup\u003e1\u003c/sup\u003eH NMR spectroscopy at 400 MHz (JNM-ECX-400 [JEOL Co. Ltd., Tokyo, Japan]) (\u003cstrong\u003eFig. S1\u003c/strong\u003e). \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta; 8.31\u0026ndash;8.01 (m, 9H), 5.84 (s, 2H), 3.65\u0026ndash;3.57 (m, 1H), 2.46\u0026ndash;2.26 (m, 2H), 1.92\u0026thinsp;\u0026minus;\u0026thinsp;0.84 (m, 33H), 0.53 (s, 3H).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.3. Synthesis of Me-LCA\u003c/h2\u003e\n \u003cp\u003eTo methylate LCA, LCA (1 mmol) was dissolved in MeOH (4.5 mL), and 4 N HCl/dioxane (0.5 mL) was mixed (\u003cstrong\u003eScheme S2\u003c/strong\u003e). The solution was stirred overnight, followed by addition of excess water. The product was precipitated. The solvent was then filtered to collect the precipitate. The precipitate was dried under vacuum and characterized by liquid-state \u003csup\u003e1\u003c/sup\u003eH NMR at 400 MHz (JNM-ECX-400 [JEOL Co. Ltd., Tokyo, Japan]) (\u003cstrong\u003eFig. S2\u003c/strong\u003e). \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta; 3.66 (s, 3H), 3.66\u0026ndash;3.58 (m, 1H), 2.39\u0026ndash;2.17 (m, 2H), 1.98\u0026thinsp;\u0026minus;\u0026thinsp;0.86 (m, 33H), 0.63 (s, 3H).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.4. Observation of lipid thin layer using fluorescence microscopy\u003c/h2\u003e\n \u003cp\u003eThin lipid layers were prepared following a method described in a previous paper\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Glass coverslips (18 mm square) were cleaned with piranha solution (95 wt% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e:30 wt% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [4:1, v/v]) for 1 h at room temperature and washed extensively with water. The coverslips were stored in a desiccator and dried. Dried coverslips were placed in an electric muffle furnace TMF-5000 (Tokyo Rikakikai Co., Ltd., Tokyo, Japan). The temperature was increased from 25\u0026deg;C to 400\u0026deg;C for 1 h (6.3 ℃/min), and the coverslips were baked at 400\u0026deg;C for 3 h. DOPC, DPPC, Chol, and Me-LCA were dissolved in chloroform (5 \u0026micro;L). The concentration was set at 20 mg/mL. Fluorescence probe solutions of Rho-DOPE in chloroform (1 mM), BODIPY-Chol in DMSO (1 mM), and Py-LCA in THF (1 mM) were prepared. The fluorescence probes were mixed at a molar ratio of 1 mol%. The mixed solution was evaporated using a rotary evaporator, and the thin lipid layer was dried overnight in a desiccator. Water (150 \u0026micro;L) was added to the thin layer, and the suspension was subjected to five freeze-thaw cycles. The suspension was dropped on a coverslip and dried for 2 d in a humidified atmosphere at 60\u0026deg;C. The coverslip was dropped on 20 vol% MeOH, and a thin lipid layer was observed using fluorescence microscopy IX-51-11FL/PH-S (Olympus Corp., Tokyo, Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.5. Preparation and observation of GUVs\u003c/h2\u003e\n \u003cp\u003eThe GUVs were prepared by gentle hydration using glucose\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. DOPC, DPPC, Chol, and Me-LCA were dissolved in chloroform (50 \u0026micro;L) and the total lipid concentration was adjusted to 1 mM. Fluorescence probe solutions, Rho-DOPE in chloroform (1 mM), BODIPY-Chol in DMSO (1 mM), and Py-LCA in THF (1 mM), were added at a molar ratio of 1 mol%. Glucose dissolved in MeOH (20 mM, 25 \u0026micro;L) was added. The solution was added to a glass test tube and the solvent was removed using a rotary evaporator. The thin lipid layer was then dried in a desiccator. The thin lipid layer was then hydrated with water (500 \u0026micro;L) and incubated for 24 h at 60\u0026deg;C. The suspension was dropped on a depression slide and observed using fluorescence microscopy (IX-51-11FL/PH-S; Olympus Corp., Tokyo, Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003e2.6. Cells and cell culture\u003c/h2\u003e\n \u003cp\u003eHep G2 cells and HeLa cells (JCRB1054 and JCRB9004; JCRB Cell Bank, Osaka, Japan) were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific; Waltham, MA, USA) in a humidified atmosphere of 5.0% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003e2.7. Synthesis of DBD-APy-LCA\u003c/h2\u003e\n \u003cp\u003eThe conjugation of (\u003cem\u003eS\u003c/em\u003e)-(+)-4-(\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (DBD-APy) with LCA was performed, as shown in \u003cstrong\u003eScheme S3\u003c/strong\u003e. DBD-APy (0.030 mmol), LCA (0.030 mmol), triethylamine (0.063 mmol), and diphenylphosphoryl azide (DPPA) (0.033 mmol) were dissolved in THF (1 mL). The solution was then stirred in the dark for 24 h. The organic solvent was removed using a rotary evaporator. The product was dried for 2\u0026ndash;3 days in a desiccator. MeOH (3 mL) was then added to the product and kept for stirring overnight. The product was centrifuged, and MeOH (1 mL) was added to the product and centrifuged. The collected product was dried for 2\u0026ndash;3 days in a desiccator. The synthesized DBD-APy-LCA was characterized by liquid-state \u003csup\u003e1\u003c/sup\u003eH NMR spectroscopy at 400 MHz (JNM-ECX-400 [JEOL Co. Ltd., Tokyo, Japan]) (\u003cstrong\u003eFig. S3\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 8.18 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.2 Hz, 1H), 7.82 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 6.19 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5 Hz, 1H), 4.46 (s, 1H), 4.41\u0026ndash;4.36 (m, 1H), 2.66 (s, 6H), 2.28\u0026ndash;2.20 (m, 2H), 2.13\u0026ndash;0.80 (m, 33H), 0.47 (s, 3H).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003e2.8. Observation of cells treated with DBD-APy-LCA using a confocal fluorescence microscopy\u003c/h2\u003e\n \u003cp\u003eHep G2 cells and HeLa cells (1.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL) were cultured in 10% FBS containing DMEM (2 mL). After incubation for 24 h in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37 ℃, the culture medium was replaced, and then 2 mL of 10% FBS containing DMEM and 5 \u0026micro;L of 7.6 mM Filipin III in DMSO was added, followed by incubation for 1 h. Then, 10 \u0026micro;L of 20 \u0026micro;M DBD-APy-LCA in DMSO and 2 \u0026micro;L of 5 mM DRAQ5 were added to the cells and incubated for 0.5 h. These cells were observed via confocal fluorescence laser microscopy (A1R+; Nikon, Tokyo, Japan) using the following filters: \u003cem\u003e\u0026lambda;\u003c/em\u003e\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;352 nm and \u003cem\u003e\u0026lambda;\u003c/em\u003e\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;461 nm for Filipin III; \u003cem\u003e\u0026lambda;\u003c/em\u003e\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;504 nm and \u003cem\u003e\u0026lambda;\u003c/em\u003e\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;511 nm for DBD-APy-LCA, and \u003cem\u003e\u0026lambda;\u003c/em\u003e\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;590\u0026ndash;595 nm and \u003cem\u003e\u0026lambda;\u003c/em\u003e\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;655\u0026ndash;660 nm for DRAQ5. Moreover, the cells were cultured for 24 h, and 1.7 mM BODIPY-Chol in DMSO was added. These cells were observed after incubation for 0.5 h via confocal fluorescence laser microscopy using the following filters: \u003cem\u003e\u0026lambda;\u003c/em\u003e\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;504 nm and \u003cem\u003e\u0026lambda;\u003c/em\u003e\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;511 nm for BODIPY-Chol.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003e2.9. Preparation of liposomes\u003c/h2\u003e\n \u003cp\u003eModel plasma membranes and liposomes were prepared using a previously described film hydration method\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The components of the liposomes (DOPC, DPPC, Chol, and Me-LCA) were dissolved in chloroform. The chloroform was removed using a rotary evaporator. After drying under vacuum overnight, the residual thin membrane was hydrated with water. The suspension was subjected to five freeze-thaw cycles and extruded to produce 100 nm diameter particles.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003ch2\u003e2.10. DPH fluorescence polarization measurement\u003c/h2\u003e\n \u003cp\u003eDPH was dissolved in THF, and its concentration was adjusted to 2.15 mM. The lipid concentration of the liposome suspensions was adjusted to 0.1 mM using water, and 1.85 \u0026micro;L of DPH solution was mixed with 10 mL liposome suspensions. The final concentrations of DPH and lipids was 0.4 \u0026micro;M and 0.1 mM (DPH:lipids\u0026thinsp;=\u0026thinsp;1:250 [mol/mol]), and the volume ratio of THF in which DPH were dissolved was less than 1 vol%. Fluorescence intensity measurements and anisotropy evaluations were performed using an RF-5300PC fluorometer (Shimadzu Corp., Kyoto, Japan) equipped with polarizing plates. DPH was excited at 360 nm, and the fluorescence intensity at 430 nm was measured. When the fluorescence intensity was measured, the temperature was maintained at 10\u0026ndash;50\u0026deg;C. The fluorescence polarization \u003cem\u003eP\u003c/em\u003evalue was calculated as follows:\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Taba\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(P=\\frac{{I}_{vv}-\\frac{{I}_{hv}}{{I}_{hh}}\\times {I}_{vh}}{{I}_{vv}+\\frac{{I}_{hv}}{{I}_{hh}}\\times {I}_{vh}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eWhere \u003cem\u003eI\u003c/em\u003e is the fluorescence intensity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, the subscripts \u003cem\u003ev\u003c/em\u003e and\u0026nbsp;\u003cem\u003eh\u003c/em\u003e indicate the orientation (vertical and horizontal) of the excitation and analyzer polarizers, respectively.\u003c/p\u003e\n \u003ch2\u003e2.11. Evaluation of hydrophobicity using fluorescence probes\u003c/h2\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003eThe fluorescence spectra of some fluorescence probes are dependent on solvents, and this phenomenon is applied to evaluate the hydrophobicity of the vesicles. Laurdan is an environmentally responsive fluorescent probe, and its hydrophobicity was evaluated at the boundary surface between the hydrophilic and hydrophobic regions of vesicles (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Laurdan was dissolved in EtOH, and the concentration was adjusted to 1 mM. The lipid concentration of the liposome suspensions was adjusted to 0.1 mM using water, and 10 \u0026micro;L of Laurdan solution was mixed with 10 mL liposome suspensions. Final concentration of Laurdan and lipids was 1 \u0026micro;M and 0.1 mM (Laurdan:lipids\u0026thinsp;=\u0026thinsp;1:100 [mol/mol]). The volume ratio of EtOH in which Laurdan was dissolved was less than 1 vol%. The fluorescence spectra of Laurdan were measured using an RF-5300PC fluorometer (Shimadzu Corp., Kyoto, Japan). Hydrophobicity was evaluated using the GP\u003csub\u003e340\u003c/sub\u003e values\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The values were calculated as follows:\u003c/div\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tabb\"\u003e\n \u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{G}\\text{P}}_{340}=\\frac{{I}_{440}-{I}_{490}}{{I}_{440}+{I}_{490}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eWhere \u003cem\u003eI\u003c/em\u003e\u003csub\u003e440\u003c/sub\u003e and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e490\u003c/sub\u003e are the fluorescence intensities at 440 and 490 nm, respectively, when Laurdan was excited at 340 nm.\u003c/p\u003e\n \u003cp\u003ePyrene is also an environmentally responsive fluorescent probe\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, and pyrene is used to evaluate the hydrophobicity of micelles\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Moreover, conjugation of dicarboxylic acids (succinic acid, suberic acid, and dodecanedioic acid) can evaluate gradual hydrophobicity in the hydrophobic region (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The pyrene-succinic acid conjugate (Py-C\u003csub\u003e3\u003c/sub\u003e-COOH), pyrene-suberic acid conjugate (Py-C\u003csub\u003e7\u003c/sub\u003e-COOH), and pyrene-dodecanedioic acid conjugate (Py-C\u003csub\u003e11\u003c/sub\u003e-COOH) were dissolved in THF, and their concentrations were adjusted to 10 mM. The solution was then mixed with chloroform to dissolve the lipids. Liposomes were prepared and adjusted to 100 nm as described above. The ratio of pyrene-dicarboxylic acid conjugates to lipids was 1:100 [mol/mol]. The prepared liposome suspension was diluted, and the final concentrations of pyrene\u0026thinsp;\u0026minus;\u0026thinsp;dicarboxylic acid conjugate and lipids were 1 \u0026micro;M and 0.1 mM. Pyrene-dicarboxylic acid conjugates were excited at 336 nm. Fluorescence spectra were measured from 350 to 600 nm at 10\u0026ndash;50\u0026deg;C using an RF-5300PC fluorometer (Shimadzu Corp., Kyoto, Japan). Hydrophobicity was evaluated based on the fluorescence intensity of peaks I (377\u0026thinsp;\u0026plusmn;\u0026thinsp;2 nm, \u003cem\u003eI\u003c/em\u003e\u003csub\u003eI\u003c/sub\u003e) and III (387\u0026thinsp;\u0026plusmn;\u0026thinsp;2 nm, \u003cem\u003eI\u003c/em\u003e\u003csub\u003eIII\u003c/sub\u003e). The ratio of \u003cem\u003eI\u003c/em\u003e\u003csub\u003eI\u003c/sub\u003e to \u003cem\u003eI\u003c/em\u003e\u003csub\u003eIII\u003c/sub\u003e (\u003cem\u003eI\u003c/em\u003e\u003csub\u003eI\u003c/sub\u003e/\u003cem\u003eI\u003c/em\u003e\u003csub\u003eIII\u003c/sub\u003e) is related to the relative permittivity of aliphatic monoalcohols, and the relative permittivity of the vesicles can be estimated from \u003cem\u003ethe I\u003c/em\u003e\u003csub\u003eI\u003c/sub\u003e/\u003cem\u003eI\u003c/em\u003e\u003csub\u003eIII\u003c/sub\u003e values based on correlation equations.\u003c/p\u003e\n \u003ch2\u003e2.12. MTT assay\u003c/h2\u003e\n \u003cp\u003eThe MTT assay was performed using the MTT Cell Count Kit (Nacalai Tesque, Kyoto, Japan). Hep G2 cells and HeLa cells were seeded on 96-well culture plates (100 \u0026micro;L, 2.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL) and cultured in DMEM supplemented with 10% FBS in an incubator. After 24 h of growth, the following formulations (3 \u0026micro;L) were added to the cells: Chol and Me-LCA diluted in PBS and treated with 200 mM M\u0026beta;CD (Sigma-Aldrich [St. Louis, MO, USA]). After incubation for 24 h, 10 \u0026micro;L MTT solution was added to the cells. After incubation for 3 h, 100 \u0026micro;L of solubilization solution was added and the formazan precipitate was dissolved by pipetting. The absorbance of each well at 570 and 650 nm was measured using a microplate spectrophotometer (Flex Station [Molecular Devices; San Jose, CA, USA]). Cell viability was calculated using the following equation:\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tabc\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{R}\\text{e}\\text{l}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e} \\text{c}\\text{e}\\text{l}\\text{l} \\text{v}\\text{i}\\text{a}\\text{b}\\text{i}\\text{l}\\text{i}\\text{t}\\text{y}=\\frac{{A}_{570}-{A}_{650}}{{{A}_{570}}_{\\left(0\\right)}-{{A}_{650}}_{\\left(0\\right)}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eWhere \u003cem\u003eA\u003c/em\u003e\u003csub\u003e570\u003c/sub\u003e and \u003cem\u003eA\u003c/em\u003e\u003csub\u003e650\u003c/sub\u003e are the absorbance values at 570 and 650 nm, respectively, when the cells were treated with the sample. \u003cem\u003eA\u003c/em\u003e\u003csub\u003e570(0)\u003c/sub\u003e and \u003cem\u003eA\u003c/em\u003e\u003csub\u003e650(0)\u003c/sub\u003e are the absorbance values at 570 and 650 nm, respectively, when the cells were treated with 200 mM M\u0026beta;CD in PBS.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Localization of Me-LCA in phospholipid layers and plasma membrane\u003c/h2\u003e \u003cp\u003eFluorescence probes, Rho-DOPE, BODIPY-Chol, and Py-LCA were mixed in thin lipid layers, and the layers were observed by fluorescence microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e(a)\u003c/b\u003e). When a thin lipid layer composed of DOPC/DPPC/Chol (1:1:1, mol/mol) was observed, the DOPC-rich L\u003csub\u003ed\u003c/sub\u003e phase and DPPC- and Chol-rich L\u003csub\u003eo\u003c/sub\u003e phases coexisted on the liposomal membrane at ~\u0026thinsp;25 ℃\u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Rho-DOPE was localized in the L\u003csub\u003ed\u003c/sub\u003e phase, whereas BODIPY-Chol was localized in the L\u003csub\u003eo\u003c/sub\u003e phase. The fluorescence of Rho-DOPE and BODIPY-Chol was observed on different parts of the thin lipid layer because of phase separation. Py-LCA fluorescence was observed in the L\u003csub\u003ed\u003c/sub\u003e phase. This result suggests that Me-LCA is localized in the L\u003csub\u003ed\u003c/sub\u003e phase, even though Me-LCA is a steroid molecule similar to Chol, which is localized in the L\u003csub\u003eo\u003c/sub\u003e phase. After excessive secretion of bile acids, they may accumulate in the plasma membrane. To investigate the behavior of Me-LCA in plasma membranes containing a large number of Me-LCA molecules, DOPC/DPPC/Chol/Me-LCA (1:1:1:1, mol/mol) lipid thin layers were also employed. The fluorescence of Rho-DOPE and Py-LCA was observed in the same part of the lipid thin layer. These fluorescence probes and BODIPY-Chol were observed in different parts of the lipid thin layer. Thus, the thin lipid layer was heterogeneous despite the presence of a large number of Me-LCA molecules (25 mol%), and Py-LCA was localized in the L\u003csub\u003ed\u003c/sub\u003e phase. Moreover, Py-LCA localization was evaluated using GUVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e(b)\u003c/b\u003e). This result using GUVs was similar to that obtained using thin lipid layers. Py-LCA was localized in the L\u003csub\u003ed\u003c/sub\u003e phase of GUVs composed of DOPC, DPPC, and Chol. Me-LCA did not affect phase separation, and Py-LCA was localized in the L\u003csub\u003ed\u003c/sub\u003e phase of GUVs composed of DOPC, DPPC, Chol, and Me-LCA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDBD-APy-LCA was also used to investigate the cellular localization of Me-LCA. DBD-APy-LCA was added to Hep G2 cells and HeLa cells, and these cells were observed by confocal fluorescence microscopy. These cells were also treated with DRAQ5 as a fluorescent DNA stain and Filipin III as a fluorescent Cholstain. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows confocal fluorescence microscopic images of Hep G2 cells and HeLa cells that were treated with these fluorescence probes. The fluorescence of DBD-APy-LCA was observed in the plasma membrane, similar to that of Filipin III. Moreover, DBD-APy-LCA was also observed in the cytoplasm. This result shows that some DBD-APy-LCA molecules are localized in the plasma membrane, whereas other molecules are localized in the cytoplasm. This result suggests that Me-LCA accumulates in the plasma membrane and the organelle membranes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Evaluation of effects of Me-LCA on membrane properties and comparison with Chol\u003c/h2\u003e \u003cp\u003eDPH fluorescence polarization measurements were employed to evaluate the microviscosity of liposomes\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e(a)\u003c/b\u003e shows the fluorescence polarization \u003cem\u003eP\u003c/em\u003e values of DOPC liposomes containing Chol and Me-LCA. The \u003cem\u003eP\u003c/em\u003e values of the DOPC liposomes increased with increasing Chol molar ratio at 10\u0026deg;C. An increase in temperature reduced the \u003cem\u003eP\u003c/em\u003e values of DOPC liposomes regardless of the Chol molar ratio, but the \u003cem\u003eP\u003c/em\u003e values of DOPC liposomes increased with increasing Chol molar ratio, independent of temperature. On the other hand, there were hardly any differences in the \u003cem\u003eP\u003c/em\u003e values regardless of the Me-LCA molar ratio at 10\u0026ndash;50\u0026deg;C. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e(b)\u003c/b\u003e shows \u003cem\u003eP\u003c/em\u003e values of DPPC liposomes. The \u003cem\u003eP\u003c/em\u003e values of DPPC liposomes were similar regardless of whether steroid molecules containing or not at \u0026le;\u0026thinsp;37 ℃. The \u003cem\u003eP\u003c/em\u003e values were different at 50\u0026deg;C for the DPPC liposomes. Although the \u003cem\u003eP\u003c/em\u003e value of DPPC liposomes without Chol remarkably decreased with a change in temperature from 37\u0026deg;C to 50\u0026deg;C, this decrease was suppressed by Chol. The smallest decrease was observed for the 50 mol% Chol. The \u003cem\u003eP\u003c/em\u003e values of DPPC liposomes with 0\u0026ndash;50 mol% Me-LCA were also measured, but no difference was observed regardless of the Me-LCA molar ratio, unlike Chol at 10\u0026ndash;50\u0026deg;C. The \u003cem\u003eP\u003c/em\u003e value of DPPC liposomes with 0 mol% Me-LCA remarkably decreased with a change in temperature from 37\u0026deg;C to 50\u0026deg;C, and the \u003cem\u003eP\u003c/em\u003e values of DPPC liposomes with 10\u0026ndash;50 mol% Me-LCA similarly changed. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e(c)\u003c/b\u003e shows the \u003cem\u003eP\u003c/em\u003e values of DOPC/DPPC/Chol (1:1:1, mol/mol) liposomes with 0\u0026ndash;25 mol% Me-LCA. The \u003cem\u003eP\u003c/em\u003e values were similar regardless of the Me-LCA molar ratio and temperature. The \u003cem\u003eP\u003c/em\u003e values decreased with increasing temperature, similar to the DOPC liposomes with Me-LCA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHydrophobicity at the boundary surface between the headgroup and acyl chain region was evaluated using a Laurdan fluorescence probe. Laurdan was used to evaluate hydrophobicity based on GP\u003csub\u003e340\u003c/sub\u003e values calculated from the fluorescence spectrum\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e(a)\u003c/b\u003e shows the GP\u003csub\u003e340\u003c/sub\u003e values of DOPC liposomes with Chol or Me-LCA. Chol increased GP\u003csub\u003e340\u003c/sub\u003e values, whereas Me-LCA did not affect GP\u003csub\u003e340\u003c/sub\u003e values in a temperature-dependent manner. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e(b)\u003c/b\u003e shows GP\u003csub\u003e340\u003c/sub\u003e values of DPPC liposomes. Differences of GP\u003csub\u003e340\u003c/sub\u003e values did not be observed regardless of whether steroid molecules containing or not at \u0026lt;\u0026thinsp;40 ℃. However, Chol increased the GP\u003csub\u003e340\u003c/sub\u003e values at 50\u0026deg;C, whereas Me-LCA did not affect GP\u003csub\u003e340\u003c/sub\u003e values. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e(c)\u003c/b\u003e shows the GP\u003csub\u003e340\u003c/sub\u003e values of DOPC/DPPC/Chol (1:1:1, mol/mol) liposomes; Me-LCA did not affect GP\u003csub\u003e340\u003c/sub\u003e values at 10\u0026ndash;50\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHydrophobicity in the hydrophobic region was also evaluated using fluorescence probes and pyrene-dicarboxylic acid conjugates (Py-C\u003csub\u003e3\u003c/sub\u003e-COOH, Py-C\u003csub\u003e7\u003c/sub\u003e-COOH, and Py-C\u003csub\u003e11\u003c/sub\u003e-COOH). Pyrene was used to evaluate hydrophobicity based on \u003cem\u003eI\u003c/em\u003e\u003csub\u003eI\u003c/sub\u003e/\u003cem\u003eI\u003c/em\u003e\u003csub\u003eIII\u003c/sub\u003e values, which are the ratio of the intensities of peak I (377\u0026thinsp;\u0026plusmn;\u0026thinsp;2 nm, \u003cem\u003eI\u003c/em\u003e\u003csub\u003eI\u003c/sub\u003e) and peak III (387\u0026thinsp;\u0026plusmn;\u0026thinsp;2 nm, \u003cem\u003eI\u003c/em\u003e\u003csub\u003eIII\u003c/sub\u003e) of the pyrene fluorescence spectrum. The \u003cem\u003eI\u003c/em\u003e\u003csub\u003eI\u003c/sub\u003e/\u003cem\u003eI\u003c/em\u003e\u003csub\u003eIII\u003c/sub\u003e values were used to estimate the relative permittivity, and Py-C\u003csub\u003e3\u003c/sub\u003e-COOH, Py-C\u003csub\u003e7\u003c/sub\u003e-COOH, and Py-C\u003csub\u003e11\u003c/sub\u003e-COOH were evaluated in the shallow, middle, and deep regions\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the estimated \u003cem\u003eε\u003c/em\u003e\u003csub\u003er\u003c/sub\u003e of the liposomes using Py-C\u003csub\u003e7\u003c/sub\u003e-COOH. The estimated \u003cem\u003eε\u003c/em\u003e\u003csub\u003er\u003c/sub\u003e of DOPC liposomes with 0\u0026ndash;50 mol% Chol was 8\u0026ndash;16 at 10\u0026ndash;50\u0026deg;C, and a remarkable difference was hardly observed, regardless of Chol. In addition, Me-LCA did not affect the estimated \u003cem\u003eε\u003c/em\u003e\u003csub\u003er\u003c/sub\u003e of the DOPC liposomes. Moreover, the estimated \u003cem\u003eε\u003c/em\u003e\u003csub\u003er\u003c/sub\u003e values of DPPC liposomes and DOPC/DPPC/Chol (1:1:1, mol/mol) liposomes were similar regardless of the molar ratio of Chol and Me-LCA. \u003cb\u003eFig. S5\u003c/b\u003e and \u003cb\u003eFig. S6\u003c/b\u003e show the estimated \u003cem\u003eε\u003c/em\u003e\u003csub\u003er\u003c/sub\u003e values of liposomes using Py-C\u003csub\u003e3\u003c/sub\u003e-COOH and Py-C\u003csub\u003e11\u003c/sub\u003e-COOH. These results also show that Me-LCA did not significantly affect the estimated \u003cem\u003eε\u003c/em\u003e\u003csub\u003er\u003c/sub\u003e of the liposomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Cytotoxicity of Me-LCA\u003c/h2\u003e \u003cp\u003eHep G2 cells and HeLa cells were treated with Chol or Me-LCA, and the cell viability of these cells was evaluated using the MTT assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). When Hep G2 cells were treated with Chol and Me-LCA at concentrations\u0026thinsp;\u0026lt;\u0026thinsp;100 \u0026micro;M, Chol and Me-LCA did not affect relative cell viability. High concentrations of Chol and Me-LCA decreased the relative cell viability. In contrast, the relative viability of HeLa cells showed different changes after treatment with Chol or Me-LCA. When HeLa cells were treated with Chol at concentrations\u0026thinsp;\u0026lt;\u0026thinsp;150 \u0026micro;M, relative cell viability increased. The relative cell viability was decreased from 2.4 to 1.2 at high concentrations. However, Me-LCA did not increase relative cell viability. Changes in the relative viability of HeLa cells treated with Me-LCA were similar to those of Hep G2 cells. Treatment with 300 \u0026micro;M Me-LCA did not affect the relative viability of HeLa cells, and a high concentration of Me-LCA decreased the relative cell viability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe cytotoxicity of bile acids is well known. A detergent model of cell death induced by bile acids posits that bile acids remove phospholipids from the plasma membrane\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In this study, we investigated the possibility that the bent structure of bile acids contributes to cell death. Me-LCA, which cannot form micelles, was used as a model bile acid. The localization of Me-LCA was initially investigated because the effects of Me-LCA on the lipid bilayer may depend on the phase state. To evaluate the effects of Me-LCA, Chol, which has a flat structure, was used. It has been referred about a relationship between localization of Chol and effects of Chol on the lipid bilayer. The condensation effect of Chol can produce many close hydrophobic contacts with the neighboring acyl chains of phospholipids, resulting in tight packing\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Moreover, Chol becomes a repulsive phospholipid as the proportion of \u003cem\u003ecis\u003c/em\u003e double bonds increases\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Thus, Chol pulls saturated phospholipids and pushes unsaturated phospholipids with \u003cem\u003ecis\u003c/em\u003e double bonds away, inducing phase separation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Other steroid molecules such as 7-dehydrocholesterol, campesterol, β-sitosterol, ergosterol, brassicasterol, and stigmasterol also increase the lipid chain order\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The condensation effect was not unique to Chol. In contrast, Me-LCA, a steroid molecule derivative, did not increase the lipid chain order. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the membrane fluidity was evaluated using DPH. Me-LCA did not affect the \u003cem\u003eP\u003c/em\u003e value, whereas Chol decreased the \u003cem\u003eP\u003c/em\u003e value. The difference between these steroid molecules containing Chol and Me-LCA is their geometrical structure. We assume that the flat structure of Chol is suitable for interaction with the neighboring acyl chains of phospholipids, but the bent structure of Me-LCA is less close to the hydrophobic contacts. This difference was also related to localization in the lipid bilayer. BODIPY-Chol localized in the L\u003csub\u003eo\u003c/sub\u003e phase, while Py-LCA localized in the L\u003csub\u003ed\u003c/sub\u003e phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This result suggests that Chol prefers saturated phospholipids to bile acids, and that Chol pushes bile acids to the L\u003csub\u003ed\u003c/sub\u003e phase.\u003c/p\u003e \u003cp\u003eWhen cells were treated with DBD-APy-LCA, fluorescence was observed in both the plasma membrane and the cytosol (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). DBD-APy-LCA is taken up into the cytosol by endocytosis and/or bile acid receptors. Filipin III binds to Chol in the plasma membrane, resulting in its fluorescence in the plasma membrane. However, when the cells were treated with BODIPY-Chol, fluorescence was observed in the cytosol (\u003cb\u003eFig. S4\u003c/b\u003e). Chol is delivered by apolipoproteins in the body, and its distribution is regulated\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In contrast, the distribution of dissolved Chol molecules in DMSO was not regulated compared with Chol in the body. Dissolved Chol molecules are distributed in the cytosol via the plasma membrane. Therefore, both dissolved steroid molecules affect the properties of the plasma membrane and those of the organelle membranes.\u003c/p\u003e \u003cp\u003eAlthough Me-LCA did not condense phospholipids, it did not perturb the lipid bilayer. When the lipid bilayer is perturbed by other molecules, such as amphiphilic peptides, the \u003cem\u003eP\u003c/em\u003e value decrease\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This perturbation promotes the penetration of water molecules into the lipid bilayer\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Thus, it suggested that the GP\u003csub\u003e340\u003c/sub\u003e value and the estimated \u003cem\u003eε\u003c/em\u003e\u003csub\u003er\u003c/sub\u003e decreased. However, Me-LCA did not affect \u003cem\u003eP\u003c/em\u003e value, GP\u003csub\u003e340\u003c/sub\u003e value, or estimated \u003cem\u003eε\u003c/em\u003e\u003csub\u003er\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results suggested that Me-LCA with a bent structure hardly perturbed the lipid bilayer. These results are related to cytotoxicity. Me-LCA did not affect proliferation of Hep G2 cells and HeLa cells. Deoxycholic acid (DC), chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA) at 750 \u0026micro;M can lyse Hep G2 cells after 4 h of incubation\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, the relative cell viability of Hep G2 cells and HeLa cells treated with Me-LCA at \u0026gt;\u0026thinsp;750 \u0026micro;M was higher than that of Hep G2 cells treated with DC, CDCA, and GCDCA at 750 \u0026micro;M. Evaluation of cytotoxicity suggested that by removing phospholipids constituting the plasma membrane, bile acid micelles mainly contribute to cell death.\u003c/p\u003e \u003cp\u003eNotably, Me-LCA cannot replace Chol. Chol is a component of raft structure, which plays an important role in regulating the behavior of membrane proteins\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. When HeLa cells were treated with Chol, their proliferation improved (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This phenomenon has been reported in human prostate cancer cells (PC-3 cells)\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. EGFR is a membrane protein that promotes cellular proliferation. The EGFR substrate 15-related protein (EPS15R) regulates the internalization of EGFR\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, resulting in the inhibition of proliferation. Chol affects EGFR internalization. Adipocyte plasma membrane-associated protein (APMAP) is a transmembrane protein that accumulates in Chol-induced lipid rafts. APMAP interacts with EPS15R. Interaction with EPS15R inhibited the internalization of EGFR and promoted proliferation.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eA methylated bile acid derivative, Me-LCA, was used to investigate the cytotoxicity induced by bile acids. Me-LCA localized in the L\u003csub\u003ed\u003c/sub\u003e phase and did not perturb the lipid bilayer. Me-LCA also did not affect cell proliferation. These results show that the removal of phospholipids from the plasma membrane is mainly responsible for the cytotoxicity induced by bile acids. Moreover, the effect of Me-LCA was compared with that of Chol, a steroid molecule similar to bile acids. Chol ordered the lipid array. Me-LCA did not perturb the membrane and order it. Chol increased the proliferation of HeLa cells, whereas Me-LCA did not. These results suggest that Me-LCA is not an alternative to Chol that shows a condensation effect and is a component of the raft structure. Me-LCA did not affect membrane properties to induce cell death.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Division of Medical Research Support of the Advanced Research Support Center at Ehime University. We would like to thank Editage (www.editage.com) for English language editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.I. wrote the main manuscript text and prepared figures 4 and 8. N.E. prepared figures 3, 5 and 7. Y.N. prepared a figure 6. T.K. synthesized and characterized Me-LCA and fluorescence probes. K.H. and H.N. are supervisors and edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated during the study are available from the corresponding author, Keita Hayashi, upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTanaka, K., Fujimura-Kamada, K. \u0026amp; Yamamoto, T. Functions of phospholipid flippases. \u003cem\u003eJ. Biochem.\u003c/em\u003e \u003cstrong\u003e149\u003c/strong\u003e, 131-143 (2010).\u003c/li\u003e\n\u003cli\u003eTang, X., Halleck, M. S., Schlegel, R. A. \u0026amp; Williamson, P. 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Hepatol.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 100325 (2021).\u003c/li\u003e\n\u003cli\u003eJiang, S.\u003cem\u003e et al.\u003c/em\u003e Cholesterol Induces Epithelial-to-Mesenchymal Transition of Prostate Cancer Cells by Suppressing Degradation of EGFR through APMAP. \u003cem\u003eCancer Res.\u003c/em\u003e \u003cstrong\u003e79\u003c/strong\u003e, 3063-3075 (2019).\u003c/li\u003e\n\u003cli\u003eCarbone, R.\u003cem\u003e et al.\u003c/em\u003e eps15 and eps15R Are Essential Components of the Endocytic Pathway. \u003cem\u003eCancer Res.\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 5498-5504 (1997).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-1700469/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1700469/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBile acids form micelles that are essential for absorbing dietary lipids. However, excessive amounts of bile acid micelles may disrupt the plasma membrane by removing phospholipids, resulting in cell death. We hypothesized that the bent geometrical structure of the steroid scaffold of bile acids decreases lipid order (similar to unsaturated phospholipids with \u003cem\u003ecis\u003c/em\u003e double bonds), disrupting the plasma membrane. Here, lithocholic acid (LCA), a bile acid, was methylated to prevent micellization. Methylated lithocholic acid (Me-LCA) was mixed with a thin phase-separated lipid layer comprising 1,2-dioleoyl-\u003cem\u003esn\u003c/em\u003e-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-\u003cem\u003esn\u003c/em\u003e-glycero-3-phosphocholine (DPPC), and cholesterol (Chol). Me-LCA was localized not in the gel phase but in the liquid crystalline phase and excess Me-LCA did not affect phase separation. Me-LCA was distributed in the plasma membrane and the organelle membranes. However, the Me-LCA did not affect the membrane properties, membrane fluidity, and hydrophobicity of liposomes composed of DOPC, DPPC and Chol, and it did not affect proliferation. These results suggest that the removal of phospholipids from the plasma membrane by bile acid micelles mainly contributes to cell death.\u003c/p\u003e","manuscriptTitle":"The Effect of Methylated Lithocholic Acid on Plasma Membrane Properties: The Difference with the Effect of Cholesterol","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-06-17 19:57:19","doi":"10.21203/rs.3.rs-1700469/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"47e00985-26e1-4b1a-b5dd-eb73dfa60659","owner":[],"postedDate":"June 17th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-09-01T06:44:21+00:00","versionOfRecord":[],"versionCreatedAt":"2022-06-17 19:57:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1700469","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1700469","identity":"rs-1700469","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}