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Enhanced Uptake of Glycerol by Butyrate Treatment in HCT-15 Human Colon Cancer Cell Line
Drug Metab. Pharmacokinet. 22 (3): 195–198 (2007).
Note Enhanced Uptake of Glycerol by Butyrate Treatment in HCT-15 Human Colon Cancer Cell Line Nami FUJIMOTO1, Katsuhisa INOUE1, Yuriko OHGUSU1, Yayoi HAYASHI2 and Hiroaki YUASA1 1Graduate
School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan 2College of Pharmacy, Kinjo Gakuin University, Nagoya, Japan
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: The HCT-15 human colon cancer cell line has a Na＋-dependent carrier-mediated transport system for the uptake of glycerol. A similar transport system has been suggested to be present also in the small intestine and is of interest with regard to its role in the absorption of glycerol and possibly some structurally related compounds. To help clarifying functional characteristics of such glycerol transport systems, we examined the eŠect of butyrate, an agent known to facilitate the diŠerentiation of cells, on glycerol uptake in HCT-15 cells. The uptake of glycerol (0.4 mM) was found to be about 5-fold greater in HCT-15 cells pretreated with butyrate (2 mM) for 24 h than in those untreated. The increase in the uptake by the butyrate treatment was due to an increase in the maximum transport rate. The eŠect of butyrate was almost completely suppressed when actinomycin D, an inhibitor of gene transcription, and cycloheximide, an inhibitor of protein synthesis, were added to the medium during the butyrate treatment. These results support the suggestion that a speciˆc carrier protein is involved in glycerol uptake by HCT-15 cells and the carrier protein is one of those inducible by butyrate-induced cell diŠerentiation.
Key words: glycerol; carrier-mediated transport; Na＋-dependence; HCT-15 cell; butyrate; induction We also found that HCT-15, a human colon cancer cell line, has a similar Na＋-dependent carrier-mediated transport system for glycerol uptake.7) Although the functional characteristics of the glycerol transport system in HCT-15 cells was not fully in agreement with those in the rat small intestine, HCT-15 could be a useful model cell line for studies to identify a group of Na＋-dependent glycerol transport systems and to elucidate their transport mechanisms. Such transport systems would be of interest as possible pathways of drug delivery and targets of drug development. Butyrate is well known as an agent that induces cell diŠerentiation and expression of various proteins, including some transporters.8–11) We here report on the enhanced glycerol uptake by butyrate treatment in HCT-15 cells, a ˆnding that suggests the involvement of a process mediated by a protein, which could be our hypothesized carrier. This ˆnding would also help in identifying a group of Na＋-dependent glycerol transport systems and elucidating their mechanisms of transport and regulation.
Introduction Glycerol is a tri-carbon polyol involved in various physiological and pathological processes as an important intermediate of energy metabolism.1) It is particularly known to be utilized for gluconeogenesis and lipogenesis. Glycerol is commonly ingested as a component of dietary fat (triglycerides) and known to be mainly absorbed as monoglycerides from the intestine after partial hydrolysis. However, a signiˆcant portion (about 20z) has been suggested to be absorbed as free glycerol liberated from monoglycerides by further hydrolysis.2) With regard to the absorption of free glycerol, it has generally been believed that paracellular passive transport is the major mechanism since it is a small hydrophilic solute. However, we recently suggested the involvement of a speciˆc carrier-mediated transport system in glycerol absorption from a series of studies using the rat small intestine.3–6) This glycerol transport system requires Na＋ and metabolic energy, and is speciˆcally inhibited by several structurally analogous compounds, such as glycerol 3-phosphate.
Received; November 21, 2006, Accepted; February 23, 2007 To whom correspondence should be addressed : Hiroaki YUASA, Ph.D., Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan. Tel. ＋81-52-836-3423, Fax. ＋81-52-836-3423, E-mail: yuasa＠phar.nagoya-cu.ac.jp
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Materials and Methods mmol) was Materials: [2-3H] glycerol (74.0 GBq W purchased from GE Healthcare Biosciences Co. (Piscataway, NJ, U.S.A.), unlabeled glycerol was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and Clear-sol II, a scintillation ‰uid, was from Nacalai Tesque, Inc. (Kyoto, Japan). All other reagents were of analytical grade and commercially available. HCT-15 cells, a human colon cancer (adenocarcinoma) cell line, were obtained from Cell Resource Center for Biomedical Research, Tohoku University. Uptake Experiments in HCT-15 Cells: HCT-15 cells were seeded at the density of 5×104 cells W well (1 mL W well) in 24-well plates (Techno Plastic Products AG, Trasadingen, Switzerland) and cultured in RPMI 1640 medium (Sigma-Aldrich Co., St. Louis, MO, U.S.A.) containing 10z FBS and 1z penicillin W streptomycin under an atmosphere of 95z air －5z CO2 at 379 C for 24 h. Then the medium was replaced with a fresh one with or without sodium butyrate (2 mM) and the cells were cultured for another 24-h period, before being used for uptake experiments. In some experiments, actinomycin D (1 mM) or cycloheximide (10 mM) was added to the medium during the period of butyrate treatment to examine if they could suppress the eŠect of butyrate on glycerol uptake. Test solutions were prepared in Hanks' solution (0.952 mM CaCl2, 5.36 mM KCl, 0.441 mM KH2 PO4, 0.821 mM MgSO4･7H2 O, 136.7 mM NaCl, 0.385 mM Na2 HPO4･12H2 O, 25 mM D-glucose, 10 mM HEPES, pH 7.5). To test solutions were added [3H]glycerol (0.4 mM) and, when required, also unlabeled glycerol to adjust the concentration. The cells in each well were preincubated in glycerol-free Hanks' solution (1 mL) for 5 min. After removing the preincubation solution, uptake was initiated by adding 0.25 mL of Hanks' solution containing [3H]glycerol at 379 C. To stop uptake, 1.5 mL of ice-cold Hanks' solution was added, and the cells were washed twice with ice-cold Hanks' solution (2 mL). To determine the amount of [3H]glycerol taken up by the cells, the cells in each well were solubilized in 0.2 M NaOH containing 0.5z SDS (0.5 mL) for 2 h, transferred to a counting vial, and then 5 mL Clear-sol II, a scintillation ‰uid, was added for liquid scintillation counting of radioactivity. The cellular protein content was determined by the method of Lowry et al.12) Data Treatment: The uptake was estimated by subtracting the amount initially adsorbed to the cells. For kinetic analysis, the uptake rate ( J ) was calculated by dividing the uptake by time during the initial uptake phase (15 min), where uptake was proportional to time, and a minor, passive (nonsaturable) component of J was subtracted to estimate the rate of uptake by carrier-
mediated (saturable) transport ( Jc ). The passive uptake component had the clearance (uptake rate W concentration) values of 0.032 and 0.15 mL W min W mg protein, respectively, as the average (n＝4) in butyrate-treated and untreated cells, being about 10z or less of the total uptake at the low concentrations where carrier-mediated transport was most e‹cient. They were estimated as the uptake clearances at a high concentration of 10 mM, where carrier-mediated transport was saturated and negligible.7) The carrier-mediated uptake of glycerol was analyzed by assuming a single Michaelis-Menten type transport component, where Jc is expressed as a function of the concentration in the medium (Cm ) and using the maximum transport rate ( Jmax ) and the Michaelis constant ( Km ) as follows:
Jc＝
Jmax Cm Km＋Cm
(1)
The kinetic parameters of Jmax and Km were estimated by ˆtting Eq. (1) to the experimental data of Jc versus Cm proˆles using a nonlinear regression program, WinNonlin (Pharsight Co., Mountain View, CA, U.S.A.), and the reciprocal of variance as the weight. The initial adsorption of glycerol to HCT-15 cells was small, typically being 0.124±0.012 mL W mg protein (mean±S.E., n＝4) as the value normalized by Cm at the Cm of 0.4 mM for regular experiments in untreated cells. This was only about 2z of glycerol uptake at 15 min. Statistical Analysis: DiŠerences between groups were examined for statistical signiˆcance by using analysis of variance (ANOVA) followed by Dunnett's test. Results and Discussion The uptake of glycerol (0.4 mM) by HCT-15 cells in 15 min was markedly (about 5 times) increased by the pretreatment of the cells with butyrate (2 mM) for 24 h, as shown in Fig. 1. This increase in glycerol uptake was almost completely suppressed when actinomycin D (1 mM), an inhibitor of gene transcription, was added to the medium during the butyrate treatment as well as when cycloheximide (10 mM), an inhibitor of protein synthesis, was added. We also observed that glycerol uptake was proportional to time during the uptake period of 15 min and further at least up to 30 min in butyrate-treated cells as well as in untreated cells (Fig. 2), indicating that an enhancement occurred in the initial uptake process. Glycerol uptake in butyratetreated cells was reduced from 11.97±0.95 pmol W min W mg protein to 0.86±0.04 pmol W min W mg protein, as the mean±S.E. (n＝4), when NaCl in the medium was replaced with mannitol and Na2 HPO4 was replaced with K2 HPO4 (Na＋-free condition), indicating almost complete inhibition (92.8z) by Na＋ removal and,
Enhanced Uptake of Glycerol by Butyrate Treatment
Fig. 1. EŠect of butyrate treatment on glycerol uptake in HCT-15 cells. Data represent the mean±S.E. (n＝4). The uptake of [3H]glycerol (0.4 mM) was evaluated at 379C and at 15 min, using cells cultured for 24 h in the presence of butyrate (2 mM), butyrate (2 mM) and actinomycin D (1 mM), or butyrate (2 mM) and cycloheximide (10 mM), or in the absence of any of these agents. The control value was 2.22 pmol W mg protein. *Signiˆcantly diŠerent from the control (a) or butyrate alone (b) at pº0.05.
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Fig. 3. EŠect of butyrate treatment on the concentration-dependent proˆle of carrier-mediated glycerol uptake in HCT-15 cells. Data represent the mean±S.E. (n＝4). The uptake of [3H]glycerol was evaluated at 379C and at 15 min, using cells cultured for 24 h in the presence of 2 mM butyrate () or in its absence (). The solid lines represent the computer-ˆtted proˆles. Table 1. EŠect of butyrate treatment on kinetic parameters of carrier-mediated glycerol uptake in HCT-15 cells Treatment
Jmax (pmol W min W mg protein)
Km ( mM)
Control Butyrate (2 mM)
20.8±3.1 100.2±9.0
23.4±4.7 45.1±7.0
Values are computer ˆtted-parameters with S.E. (n＝5 for the control and 6 for the butyrate-treated).
Fig. 2. Time courses of glycerol uptake in HCT-15 cells. Data represent the mean±S.E. (n＝8). The uptake of [3H]glycerol (0.4 mM) was evaluated at 379C, using cells cultured for 24 h in the presence of 2 mM butyrate () or in its absence ().
hence, highly Na＋-dependent nature of the uptake process. These results suggest that some protein which is induced as a result of enhanced gene transcription by butyrate is responsible for the enhanced uptake of glycerol. The induced protein is most likely our suggested Na＋-dependent carrier as the induced component of uptake was also highly Na＋-dependent. The concentration-dependent proˆles of the rate of uptake by carrier-mediated transport were well described by a single Michaelis-Menten type equation (Eq. (1)) in the butyrate-treated cells as well as in untreated cells (Fig. 3 and Table 1), suggesting that a single carrier-mediated transport system is responsible in the both. The Jmax of 100.2 pmol W min W mg protein in butyrate-treated cells was about 5-fold greater than that
of 20.8 pmol W min W mg protein in untreated cells, consistent with the suggested induction of the carrier protein by butyrate treatment. The Km of 45.1 mM in the former was also found to be greater, though to a lesser extent, than that of 23.4 mM in the latter, suggesting some reduction in a‹nity of glycerol to the carrier. However, the eŠect of alcohols on glycerol uptake in butyratetreated cells (Fig. 4) were quite consistent with that in untreated cells observed in our previous study.7) The inhibition of about 90z by 1,2-propanediol and about 60z by 1,2-ethanediol were comparable with those of about 80z and 50z, respectively, in untreated cells. Since we used the same glycerol concentration much lower than the Km in the both studies and also the same concentration of inhibitors, the similar extent of inhibition by each inhibitor suggests the a‹nity of the carrier to those potential competitive inhibitors was not altered signiˆcantly by butyrate treatment. The other alcohols which showed no or only minimal inhibition in butyrate-treated cells were not eŠective as inhibitors in untreated cells, either. Thus, although there may be some modulation in the carrier function in butyratetreated cells, as suggested by the shift in Km, it does not seem to be very critical. The passive uptake component had the clearance
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dependent glycerol transport systems, which would be of interest as possible pathways of drug delivery and targets of drug development, and elucidating their mechanisms of transport and regulation. Acknowledgments: This work was supported in part by a Grant-in-Aid for Scientiˆc Research (C) from the Japan Society for the Promotion of Science (#18590419) and by the Hoh-ansha Foundation. References 1) Fig. 4. EŠect of alcohols on glycerol uptake in butyrate-treated HCT-15 cells. Data represent the mean±S.E. (n＝4). The uptake of [3H]glycerol (0.4 mM) was evaluated at 379C and at 15 min in the presence of an alcohol (10 mM) or in its absence, using cells cultured in the presence of butyrate (2 mM) for 24 h. The control value was 14.85 pmol W mg protein. *Signiˆcantly diŠerent from the control at pº0.05.
2)
3)
4)
(uptake rate W concentration) values of 0.032±0.012 and 0.15±0.01 mL W min W mg protein, respectively, as the mean±S.E. (n＝4) in butyrate-treated and untreated cells, indicating about 5-fold lower passive transport in the former. However, at the time of uptake experiments, protein content for a well was smaller for the butyrate-treated due to slowed growth, but by 16z (0.455±0.010 and 0.540±0.011 mg protein W well, respectively, for the butyrate-treated and the untreated as the mean±S.E., n＝4), and both cells were in almost con‰uence, indicating that the cellular surface areas available for uptake for a mg protein were comparable. Therefore, the surface area is not a factor involved in the diŠerence in passive transport. Butyrate treatment might have caused some alteration in the barrier function of the cellular lipid membrane. Although passive transport is not of major interest in the present study, it may be an issue to be investigated in the future. Butyrate is well known as an agent that induces cell diŠerentiation and expression of various proteins.8–11) The mechanism of such eŠects has been traditionally been explained by inhibition of histone deacetylation, which leads to altered chromatin structure and consequently to enhanced transcription of some genes, although the mechanism of its selective action on various but limited genes has little been clariˆed. Although the detailed mechanism of butyrate action remains thus unclear, the present study has suggested that a speciˆc carrier protein is present in HCT-15 cells for glycerol uptake and it belongs to a group of carriers induced by butyrate, which include a facilitated glucose transporter,8) P-glycoprotein9,10) and Na＋ W H＋ exchan11) ger 3. This would help in identifying a group of Na＋-
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Brisson, D., Vohl, M.-C., St-Pierre, J., Hudson, T. J. and Gaudet, D.: Glycerol: a neglected variable in metabolic processes? Bioessays, 23: 534–542 (2001). Mattson, F. H. and Volpenheim, R. A.: The digestion and absorption of triglycerides. J. Biol. Chem., 239: 2772–2777 (1964). Yuasa, H., Hamamoto, K., Dogu, S., Marutani, T., Nakajima, A., Kato, T., Hayashi, Y., Inoue, K. and Watanabe, J.: Saturable absorption of glycerol in the rat intestine. Biol. Pharm. Bull., 26: 1633–1636 (2003). Kato, T., Hayashi, Y., Inoue, K. and Yuasa, H.: Functional characterization of the carrier-mediated transport system for glycerol in everted sacs of the rat small intestine. Biol. Pharm. Bull., 27: 1826–1830 (2004). Kato, T., Hayashi, Y., Inoue, K. and Yuasa, H.: Glycerol absorption by Na＋-dependent carrier-mediated transport in the closed loop of the rat small intestine. Biol. Pharm. Bull., 28: 553–555 (2005). Ohta, K., Inoue, K., Hayashi, Y. and Yuasa, H.: Carrier-mediated transport of glycerol in the perfused rat small intestine. Biol. Pharm. Bull., 29: 785–789 (2006). Fujimoto, N., Inoue, K., Hayashi, Y. and Yuasa, H.: Glycerol uptake in HCT-15 human colon cancer cell line by Na＋-dependent carrier-mediated transport. Biol. Pharm. Bull., 29: 150–154 (2006). Takano, M., Rhoads, D. B. and Isselbacher, K. J.: Sodium butyrate increases glucose transporter expression in LLC-PK1 cells. Proc. Natl. Acad. Sci. USA, 85: 8072–8075 (1988). Mickley, L. A., Bates, S. E., Richert, N. D., Currier, S., Tanaka, S., Foss, F., Rosen, N. and Fojo, A. T.: Modulation of the expression of a multidrug resistance gene ( mdr-1 W P-glycoprotein) by diŠerentiating agents. J. Biol. Chem., 264: 18031–18040 (1989). Morrow, C. S., Nakagawa, M., Goldsmith, M. E., Madden, M. J. and Cowan, K. H.: Reversible transcriptional activation of mdr1 by sodium butyrate treatment of human colon cancer cells. J. Biol. Chem., 269: 10739–10746 (1994). Kiela, P. R., Hines, E. R., Collins, J. F. and Ghishan, F. K.: Regulation of the rat NHE3 gene promoter by sodium butyrate. Am. J. Physiol., 281: G947–G956 (2001). Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randoll, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265–275 (1951).
Note Enhanced Uptake of Glycerol by Butyrate Treatment in HCT-15 Human Colon Cancer Cell Line Nami FUJIMOTO1, Katsuhisa INOUE1, Yuriko OHGUSU1, Yayoi HAYASHI2 and Hiroaki YUASA1 1Graduate
School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan 2College of Pharmacy, Kinjo Gakuin University, Nagoya, Japan
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: The HCT-15 human colon cancer cell line has a Na＋-dependent carrier-mediated transport system for the uptake of glycerol. A similar transport system has been suggested to be present also in the small intestine and is of interest with regard to its role in the absorption of glycerol and possibly some structurally related compounds. To help clarifying functional characteristics of such glycerol transport systems, we examined the eŠect of butyrate, an agent known to facilitate the diŠerentiation of cells, on glycerol uptake in HCT-15 cells. The uptake of glycerol (0.4 mM) was found to be about 5-fold greater in HCT-15 cells pretreated with butyrate (2 mM) for 24 h than in those untreated. The increase in the uptake by the butyrate treatment was due to an increase in the maximum transport rate. The eŠect of butyrate was almost completely suppressed when actinomycin D, an inhibitor of gene transcription, and cycloheximide, an inhibitor of protein synthesis, were added to the medium during the butyrate treatment. These results support the suggestion that a speciˆc carrier protein is involved in glycerol uptake by HCT-15 cells and the carrier protein is one of those inducible by butyrate-induced cell diŠerentiation.
Key words: glycerol; carrier-mediated transport; Na＋-dependence; HCT-15 cell; butyrate; induction We also found that HCT-15, a human colon cancer cell line, has a similar Na＋-dependent carrier-mediated transport system for glycerol uptake.7) Although the functional characteristics of the glycerol transport system in HCT-15 cells was not fully in agreement with those in the rat small intestine, HCT-15 could be a useful model cell line for studies to identify a group of Na＋-dependent glycerol transport systems and to elucidate their transport mechanisms. Such transport systems would be of interest as possible pathways of drug delivery and targets of drug development. Butyrate is well known as an agent that induces cell diŠerentiation and expression of various proteins, including some transporters.8–11) We here report on the enhanced glycerol uptake by butyrate treatment in HCT-15 cells, a ˆnding that suggests the involvement of a process mediated by a protein, which could be our hypothesized carrier. This ˆnding would also help in identifying a group of Na＋-dependent glycerol transport systems and elucidating their mechanisms of transport and regulation.
Introduction Glycerol is a tri-carbon polyol involved in various physiological and pathological processes as an important intermediate of energy metabolism.1) It is particularly known to be utilized for gluconeogenesis and lipogenesis. Glycerol is commonly ingested as a component of dietary fat (triglycerides) and known to be mainly absorbed as monoglycerides from the intestine after partial hydrolysis. However, a signiˆcant portion (about 20z) has been suggested to be absorbed as free glycerol liberated from monoglycerides by further hydrolysis.2) With regard to the absorption of free glycerol, it has generally been believed that paracellular passive transport is the major mechanism since it is a small hydrophilic solute. However, we recently suggested the involvement of a speciˆc carrier-mediated transport system in glycerol absorption from a series of studies using the rat small intestine.3–6) This glycerol transport system requires Na＋ and metabolic energy, and is speciˆcally inhibited by several structurally analogous compounds, such as glycerol 3-phosphate.
Received; November 21, 2006, Accepted; February 23, 2007 To whom correspondence should be addressed : Hiroaki YUASA, Ph.D., Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan. Tel. ＋81-52-836-3423, Fax. ＋81-52-836-3423, E-mail: yuasa＠phar.nagoya-cu.ac.jp
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Materials and Methods mmol) was Materials: [2-3H] glycerol (74.0 GBq W purchased from GE Healthcare Biosciences Co. (Piscataway, NJ, U.S.A.), unlabeled glycerol was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and Clear-sol II, a scintillation ‰uid, was from Nacalai Tesque, Inc. (Kyoto, Japan). All other reagents were of analytical grade and commercially available. HCT-15 cells, a human colon cancer (adenocarcinoma) cell line, were obtained from Cell Resource Center for Biomedical Research, Tohoku University. Uptake Experiments in HCT-15 Cells: HCT-15 cells were seeded at the density of 5×104 cells W well (1 mL W well) in 24-well plates (Techno Plastic Products AG, Trasadingen, Switzerland) and cultured in RPMI 1640 medium (Sigma-Aldrich Co., St. Louis, MO, U.S.A.) containing 10z FBS and 1z penicillin W streptomycin under an atmosphere of 95z air －5z CO2 at 379 C for 24 h. Then the medium was replaced with a fresh one with or without sodium butyrate (2 mM) and the cells were cultured for another 24-h period, before being used for uptake experiments. In some experiments, actinomycin D (1 mM) or cycloheximide (10 mM) was added to the medium during the period of butyrate treatment to examine if they could suppress the eŠect of butyrate on glycerol uptake. Test solutions were prepared in Hanks' solution (0.952 mM CaCl2, 5.36 mM KCl, 0.441 mM KH2 PO4, 0.821 mM MgSO4･7H2 O, 136.7 mM NaCl, 0.385 mM Na2 HPO4･12H2 O, 25 mM D-glucose, 10 mM HEPES, pH 7.5). To test solutions were added [3H]glycerol (0.4 mM) and, when required, also unlabeled glycerol to adjust the concentration. The cells in each well were preincubated in glycerol-free Hanks' solution (1 mL) for 5 min. After removing the preincubation solution, uptake was initiated by adding 0.25 mL of Hanks' solution containing [3H]glycerol at 379 C. To stop uptake, 1.5 mL of ice-cold Hanks' solution was added, and the cells were washed twice with ice-cold Hanks' solution (2 mL). To determine the amount of [3H]glycerol taken up by the cells, the cells in each well were solubilized in 0.2 M NaOH containing 0.5z SDS (0.5 mL) for 2 h, transferred to a counting vial, and then 5 mL Clear-sol II, a scintillation ‰uid, was added for liquid scintillation counting of radioactivity. The cellular protein content was determined by the method of Lowry et al.12) Data Treatment: The uptake was estimated by subtracting the amount initially adsorbed to the cells. For kinetic analysis, the uptake rate ( J ) was calculated by dividing the uptake by time during the initial uptake phase (15 min), where uptake was proportional to time, and a minor, passive (nonsaturable) component of J was subtracted to estimate the rate of uptake by carrier-
mediated (saturable) transport ( Jc ). The passive uptake component had the clearance (uptake rate W concentration) values of 0.032 and 0.15 mL W min W mg protein, respectively, as the average (n＝4) in butyrate-treated and untreated cells, being about 10z or less of the total uptake at the low concentrations where carrier-mediated transport was most e‹cient. They were estimated as the uptake clearances at a high concentration of 10 mM, where carrier-mediated transport was saturated and negligible.7) The carrier-mediated uptake of glycerol was analyzed by assuming a single Michaelis-Menten type transport component, where Jc is expressed as a function of the concentration in the medium (Cm ) and using the maximum transport rate ( Jmax ) and the Michaelis constant ( Km ) as follows:
Jc＝
Jmax Cm Km＋Cm
(1)
The kinetic parameters of Jmax and Km were estimated by ˆtting Eq. (1) to the experimental data of Jc versus Cm proˆles using a nonlinear regression program, WinNonlin (Pharsight Co., Mountain View, CA, U.S.A.), and the reciprocal of variance as the weight. The initial adsorption of glycerol to HCT-15 cells was small, typically being 0.124±0.012 mL W mg protein (mean±S.E., n＝4) as the value normalized by Cm at the Cm of 0.4 mM for regular experiments in untreated cells. This was only about 2z of glycerol uptake at 15 min. Statistical Analysis: DiŠerences between groups were examined for statistical signiˆcance by using analysis of variance (ANOVA) followed by Dunnett's test. Results and Discussion The uptake of glycerol (0.4 mM) by HCT-15 cells in 15 min was markedly (about 5 times) increased by the pretreatment of the cells with butyrate (2 mM) for 24 h, as shown in Fig. 1. This increase in glycerol uptake was almost completely suppressed when actinomycin D (1 mM), an inhibitor of gene transcription, was added to the medium during the butyrate treatment as well as when cycloheximide (10 mM), an inhibitor of protein synthesis, was added. We also observed that glycerol uptake was proportional to time during the uptake period of 15 min and further at least up to 30 min in butyrate-treated cells as well as in untreated cells (Fig. 2), indicating that an enhancement occurred in the initial uptake process. Glycerol uptake in butyratetreated cells was reduced from 11.97±0.95 pmol W min W mg protein to 0.86±0.04 pmol W min W mg protein, as the mean±S.E. (n＝4), when NaCl in the medium was replaced with mannitol and Na2 HPO4 was replaced with K2 HPO4 (Na＋-free condition), indicating almost complete inhibition (92.8z) by Na＋ removal and,
Enhanced Uptake of Glycerol by Butyrate Treatment
Fig. 1. EŠect of butyrate treatment on glycerol uptake in HCT-15 cells. Data represent the mean±S.E. (n＝4). The uptake of [3H]glycerol (0.4 mM) was evaluated at 379C and at 15 min, using cells cultured for 24 h in the presence of butyrate (2 mM), butyrate (2 mM) and actinomycin D (1 mM), or butyrate (2 mM) and cycloheximide (10 mM), or in the absence of any of these agents. The control value was 2.22 pmol W mg protein. *Signiˆcantly diŠerent from the control (a) or butyrate alone (b) at pº0.05.
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Fig. 3. EŠect of butyrate treatment on the concentration-dependent proˆle of carrier-mediated glycerol uptake in HCT-15 cells. Data represent the mean±S.E. (n＝4). The uptake of [3H]glycerol was evaluated at 379C and at 15 min, using cells cultured for 24 h in the presence of 2 mM butyrate () or in its absence (). The solid lines represent the computer-ˆtted proˆles. Table 1. EŠect of butyrate treatment on kinetic parameters of carrier-mediated glycerol uptake in HCT-15 cells Treatment
Jmax (pmol W min W mg protein)
Km ( mM)
Control Butyrate (2 mM)
20.8±3.1 100.2±9.0
23.4±4.7 45.1±7.0
Values are computer ˆtted-parameters with S.E. (n＝5 for the control and 6 for the butyrate-treated).
Fig. 2. Time courses of glycerol uptake in HCT-15 cells. Data represent the mean±S.E. (n＝8). The uptake of [3H]glycerol (0.4 mM) was evaluated at 379C, using cells cultured for 24 h in the presence of 2 mM butyrate () or in its absence ().
hence, highly Na＋-dependent nature of the uptake process. These results suggest that some protein which is induced as a result of enhanced gene transcription by butyrate is responsible for the enhanced uptake of glycerol. The induced protein is most likely our suggested Na＋-dependent carrier as the induced component of uptake was also highly Na＋-dependent. The concentration-dependent proˆles of the rate of uptake by carrier-mediated transport were well described by a single Michaelis-Menten type equation (Eq. (1)) in the butyrate-treated cells as well as in untreated cells (Fig. 3 and Table 1), suggesting that a single carrier-mediated transport system is responsible in the both. The Jmax of 100.2 pmol W min W mg protein in butyrate-treated cells was about 5-fold greater than that
of 20.8 pmol W min W mg protein in untreated cells, consistent with the suggested induction of the carrier protein by butyrate treatment. The Km of 45.1 mM in the former was also found to be greater, though to a lesser extent, than that of 23.4 mM in the latter, suggesting some reduction in a‹nity of glycerol to the carrier. However, the eŠect of alcohols on glycerol uptake in butyratetreated cells (Fig. 4) were quite consistent with that in untreated cells observed in our previous study.7) The inhibition of about 90z by 1,2-propanediol and about 60z by 1,2-ethanediol were comparable with those of about 80z and 50z, respectively, in untreated cells. Since we used the same glycerol concentration much lower than the Km in the both studies and also the same concentration of inhibitors, the similar extent of inhibition by each inhibitor suggests the a‹nity of the carrier to those potential competitive inhibitors was not altered signiˆcantly by butyrate treatment. The other alcohols which showed no or only minimal inhibition in butyrate-treated cells were not eŠective as inhibitors in untreated cells, either. Thus, although there may be some modulation in the carrier function in butyratetreated cells, as suggested by the shift in Km, it does not seem to be very critical. The passive uptake component had the clearance
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dependent glycerol transport systems, which would be of interest as possible pathways of drug delivery and targets of drug development, and elucidating their mechanisms of transport and regulation. Acknowledgments: This work was supported in part by a Grant-in-Aid for Scientiˆc Research (C) from the Japan Society for the Promotion of Science (#18590419) and by the Hoh-ansha Foundation. References 1) Fig. 4. EŠect of alcohols on glycerol uptake in butyrate-treated HCT-15 cells. Data represent the mean±S.E. (n＝4). The uptake of [3H]glycerol (0.4 mM) was evaluated at 379C and at 15 min in the presence of an alcohol (10 mM) or in its absence, using cells cultured in the presence of butyrate (2 mM) for 24 h. The control value was 14.85 pmol W mg protein. *Signiˆcantly diŠerent from the control at pº0.05.
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(uptake rate W concentration) values of 0.032±0.012 and 0.15±0.01 mL W min W mg protein, respectively, as the mean±S.E. (n＝4) in butyrate-treated and untreated cells, indicating about 5-fold lower passive transport in the former. However, at the time of uptake experiments, protein content for a well was smaller for the butyrate-treated due to slowed growth, but by 16z (0.455±0.010 and 0.540±0.011 mg protein W well, respectively, for the butyrate-treated and the untreated as the mean±S.E., n＝4), and both cells were in almost con‰uence, indicating that the cellular surface areas available for uptake for a mg protein were comparable. Therefore, the surface area is not a factor involved in the diŠerence in passive transport. Butyrate treatment might have caused some alteration in the barrier function of the cellular lipid membrane. Although passive transport is not of major interest in the present study, it may be an issue to be investigated in the future. Butyrate is well known as an agent that induces cell diŠerentiation and expression of various proteins.8–11) The mechanism of such eŠects has been traditionally been explained by inhibition of histone deacetylation, which leads to altered chromatin structure and consequently to enhanced transcription of some genes, although the mechanism of its selective action on various but limited genes has little been clariˆed. Although the detailed mechanism of butyrate action remains thus unclear, the present study has suggested that a speciˆc carrier protein is present in HCT-15 cells for glycerol uptake and it belongs to a group of carriers induced by butyrate, which include a facilitated glucose transporter,8) P-glycoprotein9,10) and Na＋ W H＋ exchan11) ger 3. This would help in identifying a group of Na＋-
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