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Functional Characteristics of Aquaporin 7 as a Facilitative Glycerol Carrier
Drug Metab. Pharmacokinet. 29 (3): 244–248 (2014).
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
Regular Article Functional Characteristics of Aquaporin 7 as a Facilitative Glycerol Carrier Takahiro K ATANO 1 , Yuko I TO 1 , Kinya O HTA 1 , Tomoya YASUJIMA 1 , Katsuhisa I NOUE 2 and Hiroaki Y UASA 1, * 1
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan 2 Department of Biopharmaceutics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: Aquaglyceroporins, which constitute a subgroup of aquaporin (AQP) water channels, had been believed to serve as channels for glycerol as well as for water. However, our recent studies have indicated that AQP9 and AQP10 operate in a carrier mode, which is of saturable nature, for glycerol transport. Assuming that such a functional characteristic could also be shared by AQP7, another aquaglyceroporin, we examined its glycerol transport function. The specific transport of glycerol by human AQP7, which was stably expressed in Madin-Darby canine kidney II cells, was indeed highly saturable, indicating the involvement of a carrier mode of operation mechanism. Kinetic analysis indicated that the specific transport conformed to Michaelis-Menten kinetics with the Michaelis constant of 11.9 µM and was not associated with a nonsaturable transport component as an indication of a simultaneous channel mode of operation, which was previously indicated for AQP10. AQP7-specific glycerol transport was furthermore found to be specifically inhibited by several compounds analogous to glycerol and operate without requiring either Na + or H + . These characteristics of the carrier mode of AQP7 operation suggest that it is a facilitative carrier for glycerol and, possibly, also for analogous compounds, providing a novel insight into its operation mechanism. Keywords: aquaporin7; carrier; facilitative transport; glycerol; fat
the carrier mode of operation was suggested to be of facilitative type and sensitive to glycerol analogs such as monoacetin and monobutyrin, indicating potential competition in substrate binding. We assumed that such a carrier mode of functional characteristic, with or without a channel mode, could also be shared by the other two aquaglyceroporins and here report our study on the glycerol transport function of AQP7, which is mainly expressed in the fat.8–11) Clarifying its functional characteristics would help further explore its potential roles in physiological processes and also in drug disposition.
Introduction Aquaglyceroporins, which constitute a subgroup of aquaporin (AQP) water channels, had been believed to serve as channels for glycerol as well as for water.1–4) Although those belonging to this subgroup of AQPs (AQP3, AQP7, AQP9 and AQP10) are of great interest for their roles in the disposition of glycerol, which is involved in various physiological and pathological processes as an intermediate of energy metabolism,5) the characteristics of their glycerol transport function have not been fully clarified, being generally assumed to operate in a channel mode, which is of nonsaturable nature, for glycerol as well as for water. However, we recently found that human AQP9, which is mainly expressed in the liver, transports glycerol in a highly saturable manner, indicating a carrier mode of operation rather than a channel mode.6) In a subsequent study on AQP10, which is mainly expressed in the small intestine, it was found to operate, more interestingly, not only in a carrier mode but also in a channel mode.7) For both the AQPs,
Materials and Methods 3
Chemicals: [2- H(N)]Glycerol (29.6 GBq/mmol) was obtained from PerkinElmer (Boston, MA) and [14C]urea (2.07 GBq/mmol) was from Moravek Biochemicals (Brea, CA). Unlabeled glycerol and urea, and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Fetal bovine serum (FBS) was obtained
Received October 29, 2013; Accepted November 25, 2013 J-STAGE Advance Published Date: December 10, 2013, doi:10.2133/dmpk.DMPK-13-RG-121 *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: [email protected] This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (25460194). 244
Facilitative Glycerol Transport by AQP7
from Invitrogen (Carlsbad, CA). All other reagents were of analytical grade and commercially available. Cell culture: Madin-Darby canine kidney (MDCK) II cells were maintained at 37°C and 5% CO2 in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Isolation of AQP7: The cDNA of human AQP7 was cloned from the human testis total RNA (Clontech, Mountain View, CA) by reverse transcription and subsequent polymerase chain reaction (PCR). In brief, a reverse transcription reaction was carried out using 3 µg of the total RNA, an oligo(dT) primer and ReverTra Ace (Toyobo, Osaka, Japan) as a reverse transcriptase. The cDNA of AQP7 was isolated from the produced cDNA mixture by PCR amplification using KOD-Plus-Neo polymerase (Toyobo) and the following primers: forward primer, 5A-AAG ATC AAG ATG CGC TGT AAC TGA G-3A; reverse primer, 5A-ATT GGG GAA TGG ATG GGA TCA C-3A. These primers were designed based on the sequence in GenBank (accession number, AB006190). PCR was performed using the following conditions: 94°C for 2 min; 33 cycles of 1) 98°C for 10 s, 2) 59°C for 30 s, and 3) 68°C for 1 min. The second PCR was performed using the PCR product as a template and a forward primer containing a XhoI restriction site (underlined), 5A-TCA CTC GAG ACA TGG TTC AAG CAT CCG-3A, and a reverse primer containing a XbaI restriction site (underlined), 5A-GTC TCT AGA TCA CAA ATA ATC TCT G-3A. PCR was performed using the following conditions: 94°C for 2 min; 33 cycles of 1) 98°C for 10 s, 2) 65°C for 30 s, and 3) 68°C for 1 min. The amplified cDNA product was digested with XhoI (Toyobo) and XbaI (Toyobo) enzymes and transferred into a mammalian expression vector, pCI-neo (Promega, Madison, WI). The sequence of the amplified cDNA product was determined with an automated sequencer (ABI PRISM 3100; Applied Biosystems, Foster City, CA) and confirmed to be identical to that in GenBank. Preparation of MDCKII cells stably expressing AQP7: MDCKII cells were transfected with the plasmid carrying the cDNA of AQP7 by using Lipofectamine 2000 (Invitrogen) as a transfection reagent, according to the manufacturer’s instructions, and cultured in DMEM supplemented with 10% FBS and 800 µg/mL geneticin for 2 to 3 weeks. Antibiotic-resistant clones were selected and tested for the transport of [3H]glycerol as a probe substrate. Mock cells were prepared by the same stable transfection procedure, using empty pCI-neo vector instead of the one carrying the cDNA of AQP7. Uptake study: MDCKII cells stably expressing AQP7 (2 © 105 cells/1 mL/well initially) were grown on 24-well plates for 48 h to confluence. The cells in each well were preincubated in 1 mL of substrate-free uptake buffer, that is, Hanks’ solution (136.7 mM NaCl, 5.36 mM KCl, 0.952 mM CaCl2, 0.812 mM MgSO4, 0.441 mM KH2PO4, 0.385 mM Na2HPO4, 25 mM D-glucose) supplemented with 10 mM HEPES (pH 7.4), for 5 min and uptake assays were started by replacing the substrate-free uptake buffer for preincubation with uptake buffer containing [3H]glycerol (0.25 mL). When the effect of pH was examined, pH was adjusted by the use of 10 mM HEPES/MES instead of 10 mM HEPES. All the procedures were conducted at 37°C. Assays were stopped by the addition of ice-cold substrate-free uptake buffer (2 mL) and the cells were washed two times with 2 mL of the same buffer. The cells were solubilized in 0.5 mL of 0.2 M NaOH solution containing 0.5% sodium dodecyl sulphate at room temperature for 1 h and the associated radioactivity was measured by liquid scintillation counting, using 3 mL of Clear-sol I (Nacalai Tesque,
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Kyoto, Japan) as a scintillation fluid, for the evaluation of uptake. Cellular protein content was determined by the BCA method (BCA Protein Assay Reagent Kit; Thermo Fisher Scientific, Waltham, MA), using bovine serum albumin as the standard. Uptake assays were also conducted in mock cells, which were transfected with empty pCI-neo vector, to estimate nonspecific uptake. The specific uptake of glycerol by AQP7 was estimated by subtracting its uptake in AQP7-expressing cells from that in mock cells. A set of experiments was also conducted to assess the transport of [14C]urea. Data analysis: The uptake rate (v) was calculated by dividing the uptake by time during the initial uptake phase (5 min), where uptake was in proportion to time. The uptake clearance (CLup) was calculated by dividing v by the substrate concentration (s). The saturable transport of glycerol was analyzed by assuming Michaelis-Menten type carrier-mediated transport, for which the CLup is described as follows: CLup = Vmax/(Km + s). The kinetic parameters of maximum transport rate (Vmax) and the Michaelis constant (Km) were estimated by fitting this equation to the experimental data profile of CLup versus s by using a nonlinear leastsquares regression analysis program, WinNonlin (Pharsight, Mountain View, CA), and the reciprocal of variance as the weight. The parameters are presented as computer-fitted ones with SE. Experimental data are presented as means « SE. Statistical analysis was performed by using Student’s t-test or, when multiple comparisons were needed, ANOVA followed by Dunnett’s test, with p < 0.05 considered significant. Results Kinetic characteristics of glycerol transport: We first examined the time course of the uptake of glycerol at a trace concentration of 0.05 µM. As shown in Figure 1, it was much greater in MDCKII cells stably expressing AQP7 than in mock cells, indicating that AQP7 is highly capable of transporting glycerol, and increased in proportion to time up to 20 min. Within that time range, we set a 5-min uptake period for the evaluation of transport in the initial uptake phase in subsequent experiments. The concentration dependence of the specific uptake of glycerol by AQP7 was then examined to assess the saturability of transport. As indicated by the decline in the uptake clearance with an increase in concentration (Fig. 2), the AQP7-specific uptake was found to be highly saturable and, hence, could be assumed to be mediated by a carrier mode of operation. It is notable that the uptake clearance was reduced to an undetectable level at high concentrations,
Fig. 1. Time course of glycerol uptake in MDCKII cells stably expressing AQP7 The uptake of [3H]glycerol (0.05 µM) was evaluated at 37°C and pH 7.4. Data are presented as means « SE (n = 3).
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Fig. 2. Concentration dependence of glycerol uptake by AQP7 stably expressed in MDCKII cells The specific uptake of [3H]glycerol was evaluated for 5 min at 37°C and pH 7.4. The Vmax and Km are 406 « 33 pmol/min/mg protein and 11.9 « 1.2 µM, respectively, as the computer-fitted parameters with SE. Data are presented as means « SE (n = 3).
Fig. 4. Effect of ions on glycerol uptake by AQP7 stably expressed in MDCKII cells The specific uptake of [3H]glycerol (0.05 µM) was evaluated for 5 min at 37°C and pH 7.4. NaCl in the control medium was replaced as indicated. The control value was 1.03 pmol/min/mg protein. Data are presented as means « SE (n = 3).
Fig. 3. Time course of urea uptake in MDCKII cells stably expressing AQP7 The uptake of [14C]urea (0.7 µM) was evaluated at 37°C and pH 7.4. Data are presented as means « SE (n = 4).
Fig. 5. Effect of pH on glycerol uptake by AQP7 stably expressed in MDCKII cells The specific uptake of [3H]glycerol (0.05 µM) was evaluated for 5 min at 37°C. Data are presented as means « SE (n = 3). *p < 0.05 compared with the values at all the other pH’s.
suggesting that nonsaturable transport by a channel mode of operation is not involved. According to kinetic analysis, the concentration-dependent profile of the uptake clearance conformed to the Michaelis-Menten kinetics with the Km of 11.9 µM. We also examined the uptake of urea, which was previously indicated to be a substrate for the channel mode, but not the carrier mode, of operation of AQP10,7) for comparison. However, its uptake evaluated at the trace concentration of 0.7 µM was not altered by the introduction of AQP7 to MDCKII cells (Fig. 3), suggesting that this solute is unlikely to be a substrate of AQP7. It seems to be consistent with the finding that AQP7 does not have a channel mode for glycerol transport, which would be, if present, likely to operate also for urea transport. Effects of ionic conditions and temperature on glycerol transport: The specific uptake of glycerol by AQP7 was not altered when NaCl in the medium was replaced with KCl, choline Cl or mannitol (Fig. 4), suggesting that Na+ is not required for the carrier mode of AQP7 operation. The absence of the effect of the replacement of NaCl with mannitol to eliminate Cl¹ as well as Na+ indicates that Cl¹ is not required, either. The replacement of NaCl with Na gluconate and K gluconate to introduce gluconate, replacing Cl¹, caused moderate, though statistically insignificant, reduction in the AQP7-specific uptake. It may suggest some sensitivity of AQP7 to gluconate rather than Cl¹ requirement in AQP7 operation. At least Cl¹ is not apparently an element required for AQP7 operation.
It was also found that the AQP7-specific uptake is not dependent on pH in the range of pH 5.5 to 8, as shown in Figure 5. Although the uptake was reduced at the lowest pH of 5, it may not be very meaningful physiologically and, hence, it is most likely that H+ is not required for AQP7 operation, either. Thus, none of Na+, H+ or Cl¹, which are major ions typically involved in secondary active transport, was suggested to be required for the carrier mode of AQP7 operation. However, the AQP7-specific uptake was highly sensitive to temperature, being reduced significantly when temperature was lowered from 37°C to 4°C (Fig. 6), as additional evidence that supports the carrier mode of operation mechanism. Based on all these results, it is most likely that the carrier mode of AQP7 operation is of facilitative type. Effect of various compounds on glycerol transport: The specific uptake of glycerol by AQP7 was found to be specifically inhibited by several compounds analogous to glycerol. As shown in Figure 7, monoacetin and monobutyrin, which are glycerol esters, and diglycerol, which is an ether type of glycerol derivative, inhibited the AQP7-specific glycerol uptake almost completely at their concentration of 10 mM. Chloramphenicol, a drug having a glycerol-like moiety in its molecule, also did, though a little less potently. However, urea, which is not structurally related to glycerol, did not, consistent with the finding that it is not transported by AQP7 (Fig. 3) and, hence, at least it cannot be a competitive inhibitor.
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
Facilitative Glycerol Transport by AQP7
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others in having a 1,2-diol structure with a 2- or 3-carbon backbone. With the only exception of chloramphenicol, all those alcohols which inhibited the AQP7-specific uptake shown in Figure 7 also have the same structural characteristic. Therefore, the 1,2-diol structure with a 2 or 3-carbon backbone may play an important role in recognition by AQP7, and it may be possible that they are also transported by the carrier mode of AQP7 operation. Discussion
Fig. 6. Effect of temperature on glycerol uptake by AQP7 stably expressed in MDCKII cells The specific uptake of [3H]glycerol (0.05 µM) was evaluated for 5 min at pH 7.4. Data are presented as means « SE (n = 3). *p < 0.05 compared with the value at 37°C.
Fig. 7. Effect of various compounds on glycerol uptake by AQP7 stably expressed in MDCKII cells The specific uptake of [3H]glycerol (0.05 µM) was evaluated for 5 min at 37°C and pH 7.4 in the presence of a test compound (10 mM) or in its absence. The control value was 0.66 pmol/min/mg protein. Data are presented as means « SE (n = 3). *p < 0.05 compared with the control.
Fig. 8. Effect of various alcohols on glycerol uptake by AQP7 stably expressed in MDCKII cells The specific uptake of [3H]glycerol (0.05 µM) was evaluated for 5 min at 37°C and pH 7.4 in the presence of an alcohol (10 mM) or in its absence. The control value was 0.55 pmol/min/mg protein. Data are presented as means « SE (n = 3). *p < 0.05 compared with the control.
We also examined the effect of a group of alcohols on the specific uptake of glycerol by AQP7, as shown in Figure 8. Among them, only 1,2-ethanediol and 1,2-propanediol were found to inhibit the AQP7-specific uptake. It is also notable that S-1,2propanediol was more potent in inhibition than R-1,2-propanediol, suggesting enantioselectivity in recognition by AQP7. The two inhibiting alcohols are polyols more analogous to glycerol than the
We were successful in unveiling the carrier mode of AQP7 operation, a functional characteristic which it shares with AQP9 and AQP10.6,7) However, a simultaneous channel mode of operation, which was previously observed for AQP10 but not for AQP9, was not observed for AQP7. Therefore, this fat-specific AQP was suggested to be more analogous to liver-specific AQP9 than intestine-specific AQP10 in that kinetic characteristic. In earlier studies from other research groups, however, AQP7 was suggested to be able to transport urea as well as glycerol.8,9) The reason for this discrepancy about urea transport is unknown at this time and remains to be resolved in the future. The Km of AQP7 for glycerol (11.9 µM) is comparable with those of AQP9 (9.2 µM) and AQP10 (10.4 µM),6,7) and is relatively close to the glycerol level in the systemic circulation, which could be several tens of µM or greater (up to several hundreds of µM).12) Therefore, it could be assumed that the carrier mode of operation provides a mechanism to restrict the cellular uptake of glycerol, responding to glycerol concentration in plasma. In adipocytes, the major role of AQP7 has been suggested to be its operation for the release of glycerol generated by lipolysis in the cells to plasma.10,11) Since AQP7 has been suggested to be a facilitative carrier, which does not require any major ion for operation, it could also operate for the process of glycerol release. When starved, AQP7 is known to be upregulated in adipocytes to enhance the release of glycerol to increase its supply to the liver, where it is utilized for gluconeogenesis as a mechanism to produce glucose for the survival of the living body.13) More direct evidence for the role of AQP7 in glycerol release in adipocytes has been provided by a study using AQP7-knockout mice,14) in which glycerol was shown to be accumulated more in the knockout mice than in wild-type mice and released more slowly from adipocytes in the former than the latter. Our suggested carrier mode of its operation could be an additional mechanism to adjust glycerol release. Because glycerol concentration is supposed to be greater in adipocytes than in plasma when it is released, AQP7-mediated transport could be almost saturated and, hence, the release rate may be mainly controlled by the maximum transport rate and the number of AQP7 molecules. The characteristics of the carrier mode of AQP7 operation are also quite similar to those of AQP9 and AQP10 in that it is of facilitative type and all the inhibitors are similarly effective. In our previous studies,6,7) AQP9 and AQP10 were found not to require Na+ for their carrier mode of glycerol transport function and, hence, assumed to be facilitative carriers. For AQP10, the pHinsensitive characteristic was also confirmed. In addition, glycerol transports by these AQPs were inhibited extensively by monoacetin, monobutyrin, diglycerol and 1,2-propanediol at their concentration of 10 mM, and, to a lesser extent, by 1,2-ethanediol. Moreover, S-1,2-propanediol was more potent in inhibiting them than R-1,2-propanediol, manifesting the enantioselective characterisitc. Thus, all these characteristics of AQP9 and AQP10 are
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
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quite similar to those of AQP7 observed in the present study. The only notable difference is that AQP7 is more sensitive to chloramphenicol than AQP9 and AQP10. Whereas AQP7-specific glycerol transport was inhibited by about 85% by this drug (10 mM), the AQP9-specific one was inhibited only modestly by about 30%6) and the AQP10-specific one was not inhibited at all.7) In conclusion, AQP7 was found to function as a facilitative carrier for glycerol and, possibly, also for analogous compounds. This finding redefines the functional characteristics of this protein, which has been implicitly presumed to function as a channel for glycerol as well as for water as a member of AQP water channels, and provides a novel insight into its operation mechanism. This insight will help further explore its potential roles in physiological processes and also in drug disposition.
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Agre, P., Bonhivers, M. and Borgnia, M. J.: The aquaporins, blueprints for cellular plumbing systems. J. Biol. Chem., 273: 14659–14662 (1998). Gomes, D., Agasse, A., Thiébaud, P., Delrot, S., Gerós, H. and Chaumont, F.: Aquaporins are multifunctional water and solute transporters highly divergent in living organisms. Biochim. Biophys. Acta, 1788: 1213–1228 (2009). Hibuse, T., Maeda, N., Nagasawa, A. and Funahashi, T.: Aquaporins and glycerol metabolism. Biochim. Biophys. Acta, 1758: 1004–1011 (2006). Morishita, Y., Sakube, Y., Sasaki, S. and Ishibashi, K.: Molecular mechanisms and drug development in aquaporin water channel diseases: aquaporin superfamily (superaquaporins): expansion of aquaporins restricted to multicellular organisms. J. Pharmacol. Sci., 96: 276–279 (2004). 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).
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Ohgusu, Y., Ohta, K., Ishii, M., Katano, T., Urano, K., Watanabe, J., Inoue, K. and Yuasa, H.: Functional characterization of human aquaporin 9 as a facilitative glycerol carrier. Drug Metab. Pharmacokinet., 23: 279– 284 (2008). Ishii, M., Ohta, K., Katano, T., Urano, K., Watanabe, J., Miyamoto, A., Inoue, K. and Yuasa, H.: Dual functional characteristic of human aquaporin 10 for solute transport. Cell. Physiol. Biochem., 27: 749–756 (2011). Ishibashi, K., Kuwahara, M., Gu, Y., Kageyama, Y., Tohsaka, A., Suzuki, F., Marumo, F. and Sasaki, S.: Cloning and functional expression of a new water channel abundantly expressed in the testis permeable to water, glycerol, and urea. J. Biol. Chem., 272: 20782–20786 (1997). Kuriyama, H., Kawamoto, S., Ishida, N., Ohno, I., Mita, S., Matsuzawa, Y., Matsubara, K. and Okubo, K.: Molecular cloning and expression of a novel human aquaporin from adipose tissue with glycerol permeability. Biochem. Biophys. Res. Commun., 241: 53–58 (1997). Kishida, K., Kuriyama, H., Funahashi, T., Shimomura, I., Kihara, S., Ouchi, N., Nishida, M., Nishizawa, H., Matsuda, M., Takahashi, M., Hotta, K., Nakamura, T., Yamashita, S., Tochino, Y. and Matsuzawa, Y.: Aquaporin adipose, a putative glycerol channel in adipocytes. J. Biol. Chem., 275: 20896–20902 (2000). Kishida, K., Shimomura, I., Kondo, H., Kuriyama, H., Makino, Y., Nishizawa, H., Maeda, N., Matsuda, M., Ouchi, N., Kihara, S., Kurachi, Y., Funahashi, T. and Matsuzawa, Y.: Genomic structure and insulinmediated repression of the aquaporin adipose (AQPap), adipose-specific glycerol channel. J. Biol. Chem., 276: 36251–36260 (2001). Robergs, R. A. and Griffin, S. E.: Glycerol: biochemistry, pharmacokinetics and clinical and practical applications. Sports Med., 26: 145–167 (1998). Kuriyama, H., Shimomura, I., Kishida, K., Kondo, H., Furuyama, N., Nishizawa, H., Maeda, N., Matsuda, M., Nagaretani, H., Kihara, S., Nakamura, T., Tochino, Y., Funahashi, T. and Matsuzawa, Y.: Coordinated regulation of fat-specific and liver-specific glycerol channels, aquaporin adipose and aquaporin 9. Diabetes, 51: 2915–2921 (2002). Hara-Chikuma, M., Sohara, E., Rai, T., Ikawa, M., Okabe, M., Sasaki, S., Uchida, S. and Verkman, A. S.: Progressive adipocyte hypertrophy in aquaporin-7-deficient mice. J. Biol. Chem., 280: 15493–15496 (2005).
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
Regular Article Functional Characteristics of Aquaporin 7 as a Facilitative Glycerol Carrier Takahiro K ATANO 1 , Yuko I TO 1 , Kinya O HTA 1 , Tomoya YASUJIMA 1 , Katsuhisa I NOUE 2 and Hiroaki Y UASA 1, * 1
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan 2 Department of Biopharmaceutics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: Aquaglyceroporins, which constitute a subgroup of aquaporin (AQP) water channels, had been believed to serve as channels for glycerol as well as for water. However, our recent studies have indicated that AQP9 and AQP10 operate in a carrier mode, which is of saturable nature, for glycerol transport. Assuming that such a functional characteristic could also be shared by AQP7, another aquaglyceroporin, we examined its glycerol transport function. The specific transport of glycerol by human AQP7, which was stably expressed in Madin-Darby canine kidney II cells, was indeed highly saturable, indicating the involvement of a carrier mode of operation mechanism. Kinetic analysis indicated that the specific transport conformed to Michaelis-Menten kinetics with the Michaelis constant of 11.9 µM and was not associated with a nonsaturable transport component as an indication of a simultaneous channel mode of operation, which was previously indicated for AQP10. AQP7-specific glycerol transport was furthermore found to be specifically inhibited by several compounds analogous to glycerol and operate without requiring either Na + or H + . These characteristics of the carrier mode of AQP7 operation suggest that it is a facilitative carrier for glycerol and, possibly, also for analogous compounds, providing a novel insight into its operation mechanism. Keywords: aquaporin7; carrier; facilitative transport; glycerol; fat
the carrier mode of operation was suggested to be of facilitative type and sensitive to glycerol analogs such as monoacetin and monobutyrin, indicating potential competition in substrate binding. We assumed that such a carrier mode of functional characteristic, with or without a channel mode, could also be shared by the other two aquaglyceroporins and here report our study on the glycerol transport function of AQP7, which is mainly expressed in the fat.8–11) Clarifying its functional characteristics would help further explore its potential roles in physiological processes and also in drug disposition.
Introduction Aquaglyceroporins, which constitute a subgroup of aquaporin (AQP) water channels, had been believed to serve as channels for glycerol as well as for water.1–4) Although those belonging to this subgroup of AQPs (AQP3, AQP7, AQP9 and AQP10) are of great interest for their roles in the disposition of glycerol, which is involved in various physiological and pathological processes as an intermediate of energy metabolism,5) the characteristics of their glycerol transport function have not been fully clarified, being generally assumed to operate in a channel mode, which is of nonsaturable nature, for glycerol as well as for water. However, we recently found that human AQP9, which is mainly expressed in the liver, transports glycerol in a highly saturable manner, indicating a carrier mode of operation rather than a channel mode.6) In a subsequent study on AQP10, which is mainly expressed in the small intestine, it was found to operate, more interestingly, not only in a carrier mode but also in a channel mode.7) For both the AQPs,
Materials and Methods 3
Chemicals: [2- H(N)]Glycerol (29.6 GBq/mmol) was obtained from PerkinElmer (Boston, MA) and [14C]urea (2.07 GBq/mmol) was from Moravek Biochemicals (Brea, CA). Unlabeled glycerol and urea, and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Fetal bovine serum (FBS) was obtained
Received October 29, 2013; Accepted November 25, 2013 J-STAGE Advance Published Date: December 10, 2013, doi:10.2133/dmpk.DMPK-13-RG-121 *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: [email protected] This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (25460194). 244
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from Invitrogen (Carlsbad, CA). All other reagents were of analytical grade and commercially available. Cell culture: Madin-Darby canine kidney (MDCK) II cells were maintained at 37°C and 5% CO2 in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Isolation of AQP7: The cDNA of human AQP7 was cloned from the human testis total RNA (Clontech, Mountain View, CA) by reverse transcription and subsequent polymerase chain reaction (PCR). In brief, a reverse transcription reaction was carried out using 3 µg of the total RNA, an oligo(dT) primer and ReverTra Ace (Toyobo, Osaka, Japan) as a reverse transcriptase. The cDNA of AQP7 was isolated from the produced cDNA mixture by PCR amplification using KOD-Plus-Neo polymerase (Toyobo) and the following primers: forward primer, 5A-AAG ATC AAG ATG CGC TGT AAC TGA G-3A; reverse primer, 5A-ATT GGG GAA TGG ATG GGA TCA C-3A. These primers were designed based on the sequence in GenBank (accession number, AB006190). PCR was performed using the following conditions: 94°C for 2 min; 33 cycles of 1) 98°C for 10 s, 2) 59°C for 30 s, and 3) 68°C for 1 min. The second PCR was performed using the PCR product as a template and a forward primer containing a XhoI restriction site (underlined), 5A-TCA CTC GAG ACA TGG TTC AAG CAT CCG-3A, and a reverse primer containing a XbaI restriction site (underlined), 5A-GTC TCT AGA TCA CAA ATA ATC TCT G-3A. PCR was performed using the following conditions: 94°C for 2 min; 33 cycles of 1) 98°C for 10 s, 2) 65°C for 30 s, and 3) 68°C for 1 min. The amplified cDNA product was digested with XhoI (Toyobo) and XbaI (Toyobo) enzymes and transferred into a mammalian expression vector, pCI-neo (Promega, Madison, WI). The sequence of the amplified cDNA product was determined with an automated sequencer (ABI PRISM 3100; Applied Biosystems, Foster City, CA) and confirmed to be identical to that in GenBank. Preparation of MDCKII cells stably expressing AQP7: MDCKII cells were transfected with the plasmid carrying the cDNA of AQP7 by using Lipofectamine 2000 (Invitrogen) as a transfection reagent, according to the manufacturer’s instructions, and cultured in DMEM supplemented with 10% FBS and 800 µg/mL geneticin for 2 to 3 weeks. Antibiotic-resistant clones were selected and tested for the transport of [3H]glycerol as a probe substrate. Mock cells were prepared by the same stable transfection procedure, using empty pCI-neo vector instead of the one carrying the cDNA of AQP7. Uptake study: MDCKII cells stably expressing AQP7 (2 © 105 cells/1 mL/well initially) were grown on 24-well plates for 48 h to confluence. The cells in each well were preincubated in 1 mL of substrate-free uptake buffer, that is, Hanks’ solution (136.7 mM NaCl, 5.36 mM KCl, 0.952 mM CaCl2, 0.812 mM MgSO4, 0.441 mM KH2PO4, 0.385 mM Na2HPO4, 25 mM D-glucose) supplemented with 10 mM HEPES (pH 7.4), for 5 min and uptake assays were started by replacing the substrate-free uptake buffer for preincubation with uptake buffer containing [3H]glycerol (0.25 mL). When the effect of pH was examined, pH was adjusted by the use of 10 mM HEPES/MES instead of 10 mM HEPES. All the procedures were conducted at 37°C. Assays were stopped by the addition of ice-cold substrate-free uptake buffer (2 mL) and the cells were washed two times with 2 mL of the same buffer. The cells were solubilized in 0.5 mL of 0.2 M NaOH solution containing 0.5% sodium dodecyl sulphate at room temperature for 1 h and the associated radioactivity was measured by liquid scintillation counting, using 3 mL of Clear-sol I (Nacalai Tesque,
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Kyoto, Japan) as a scintillation fluid, for the evaluation of uptake. Cellular protein content was determined by the BCA method (BCA Protein Assay Reagent Kit; Thermo Fisher Scientific, Waltham, MA), using bovine serum albumin as the standard. Uptake assays were also conducted in mock cells, which were transfected with empty pCI-neo vector, to estimate nonspecific uptake. The specific uptake of glycerol by AQP7 was estimated by subtracting its uptake in AQP7-expressing cells from that in mock cells. A set of experiments was also conducted to assess the transport of [14C]urea. Data analysis: The uptake rate (v) was calculated by dividing the uptake by time during the initial uptake phase (5 min), where uptake was in proportion to time. The uptake clearance (CLup) was calculated by dividing v by the substrate concentration (s). The saturable transport of glycerol was analyzed by assuming Michaelis-Menten type carrier-mediated transport, for which the CLup is described as follows: CLup = Vmax/(Km + s). The kinetic parameters of maximum transport rate (Vmax) and the Michaelis constant (Km) were estimated by fitting this equation to the experimental data profile of CLup versus s by using a nonlinear leastsquares regression analysis program, WinNonlin (Pharsight, Mountain View, CA), and the reciprocal of variance as the weight. The parameters are presented as computer-fitted ones with SE. Experimental data are presented as means « SE. Statistical analysis was performed by using Student’s t-test or, when multiple comparisons were needed, ANOVA followed by Dunnett’s test, with p < 0.05 considered significant. Results Kinetic characteristics of glycerol transport: We first examined the time course of the uptake of glycerol at a trace concentration of 0.05 µM. As shown in Figure 1, it was much greater in MDCKII cells stably expressing AQP7 than in mock cells, indicating that AQP7 is highly capable of transporting glycerol, and increased in proportion to time up to 20 min. Within that time range, we set a 5-min uptake period for the evaluation of transport in the initial uptake phase in subsequent experiments. The concentration dependence of the specific uptake of glycerol by AQP7 was then examined to assess the saturability of transport. As indicated by the decline in the uptake clearance with an increase in concentration (Fig. 2), the AQP7-specific uptake was found to be highly saturable and, hence, could be assumed to be mediated by a carrier mode of operation. It is notable that the uptake clearance was reduced to an undetectable level at high concentrations,
Fig. 1. Time course of glycerol uptake in MDCKII cells stably expressing AQP7 The uptake of [3H]glycerol (0.05 µM) was evaluated at 37°C and pH 7.4. Data are presented as means « SE (n = 3).
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Fig. 2. Concentration dependence of glycerol uptake by AQP7 stably expressed in MDCKII cells The specific uptake of [3H]glycerol was evaluated for 5 min at 37°C and pH 7.4. The Vmax and Km are 406 « 33 pmol/min/mg protein and 11.9 « 1.2 µM, respectively, as the computer-fitted parameters with SE. Data are presented as means « SE (n = 3).
Fig. 4. Effect of ions on glycerol uptake by AQP7 stably expressed in MDCKII cells The specific uptake of [3H]glycerol (0.05 µM) was evaluated for 5 min at 37°C and pH 7.4. NaCl in the control medium was replaced as indicated. The control value was 1.03 pmol/min/mg protein. Data are presented as means « SE (n = 3).
Fig. 3. Time course of urea uptake in MDCKII cells stably expressing AQP7 The uptake of [14C]urea (0.7 µM) was evaluated at 37°C and pH 7.4. Data are presented as means « SE (n = 4).
Fig. 5. Effect of pH on glycerol uptake by AQP7 stably expressed in MDCKII cells The specific uptake of [3H]glycerol (0.05 µM) was evaluated for 5 min at 37°C. Data are presented as means « SE (n = 3). *p < 0.05 compared with the values at all the other pH’s.
suggesting that nonsaturable transport by a channel mode of operation is not involved. According to kinetic analysis, the concentration-dependent profile of the uptake clearance conformed to the Michaelis-Menten kinetics with the Km of 11.9 µM. We also examined the uptake of urea, which was previously indicated to be a substrate for the channel mode, but not the carrier mode, of operation of AQP10,7) for comparison. However, its uptake evaluated at the trace concentration of 0.7 µM was not altered by the introduction of AQP7 to MDCKII cells (Fig. 3), suggesting that this solute is unlikely to be a substrate of AQP7. It seems to be consistent with the finding that AQP7 does not have a channel mode for glycerol transport, which would be, if present, likely to operate also for urea transport. Effects of ionic conditions and temperature on glycerol transport: The specific uptake of glycerol by AQP7 was not altered when NaCl in the medium was replaced with KCl, choline Cl or mannitol (Fig. 4), suggesting that Na+ is not required for the carrier mode of AQP7 operation. The absence of the effect of the replacement of NaCl with mannitol to eliminate Cl¹ as well as Na+ indicates that Cl¹ is not required, either. The replacement of NaCl with Na gluconate and K gluconate to introduce gluconate, replacing Cl¹, caused moderate, though statistically insignificant, reduction in the AQP7-specific uptake. It may suggest some sensitivity of AQP7 to gluconate rather than Cl¹ requirement in AQP7 operation. At least Cl¹ is not apparently an element required for AQP7 operation.
It was also found that the AQP7-specific uptake is not dependent on pH in the range of pH 5.5 to 8, as shown in Figure 5. Although the uptake was reduced at the lowest pH of 5, it may not be very meaningful physiologically and, hence, it is most likely that H+ is not required for AQP7 operation, either. Thus, none of Na+, H+ or Cl¹, which are major ions typically involved in secondary active transport, was suggested to be required for the carrier mode of AQP7 operation. However, the AQP7-specific uptake was highly sensitive to temperature, being reduced significantly when temperature was lowered from 37°C to 4°C (Fig. 6), as additional evidence that supports the carrier mode of operation mechanism. Based on all these results, it is most likely that the carrier mode of AQP7 operation is of facilitative type. Effect of various compounds on glycerol transport: The specific uptake of glycerol by AQP7 was found to be specifically inhibited by several compounds analogous to glycerol. As shown in Figure 7, monoacetin and monobutyrin, which are glycerol esters, and diglycerol, which is an ether type of glycerol derivative, inhibited the AQP7-specific glycerol uptake almost completely at their concentration of 10 mM. Chloramphenicol, a drug having a glycerol-like moiety in its molecule, also did, though a little less potently. However, urea, which is not structurally related to glycerol, did not, consistent with the finding that it is not transported by AQP7 (Fig. 3) and, hence, at least it cannot be a competitive inhibitor.
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
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others in having a 1,2-diol structure with a 2- or 3-carbon backbone. With the only exception of chloramphenicol, all those alcohols which inhibited the AQP7-specific uptake shown in Figure 7 also have the same structural characteristic. Therefore, the 1,2-diol structure with a 2 or 3-carbon backbone may play an important role in recognition by AQP7, and it may be possible that they are also transported by the carrier mode of AQP7 operation. Discussion
Fig. 6. Effect of temperature on glycerol uptake by AQP7 stably expressed in MDCKII cells The specific uptake of [3H]glycerol (0.05 µM) was evaluated for 5 min at pH 7.4. Data are presented as means « SE (n = 3). *p < 0.05 compared with the value at 37°C.
Fig. 7. Effect of various compounds on glycerol uptake by AQP7 stably expressed in MDCKII cells The specific uptake of [3H]glycerol (0.05 µM) was evaluated for 5 min at 37°C and pH 7.4 in the presence of a test compound (10 mM) or in its absence. The control value was 0.66 pmol/min/mg protein. Data are presented as means « SE (n = 3). *p < 0.05 compared with the control.
Fig. 8. Effect of various alcohols on glycerol uptake by AQP7 stably expressed in MDCKII cells The specific uptake of [3H]glycerol (0.05 µM) was evaluated for 5 min at 37°C and pH 7.4 in the presence of an alcohol (10 mM) or in its absence. The control value was 0.55 pmol/min/mg protein. Data are presented as means « SE (n = 3). *p < 0.05 compared with the control.
We also examined the effect of a group of alcohols on the specific uptake of glycerol by AQP7, as shown in Figure 8. Among them, only 1,2-ethanediol and 1,2-propanediol were found to inhibit the AQP7-specific uptake. It is also notable that S-1,2propanediol was more potent in inhibition than R-1,2-propanediol, suggesting enantioselectivity in recognition by AQP7. The two inhibiting alcohols are polyols more analogous to glycerol than the
We were successful in unveiling the carrier mode of AQP7 operation, a functional characteristic which it shares with AQP9 and AQP10.6,7) However, a simultaneous channel mode of operation, which was previously observed for AQP10 but not for AQP9, was not observed for AQP7. Therefore, this fat-specific AQP was suggested to be more analogous to liver-specific AQP9 than intestine-specific AQP10 in that kinetic characteristic. In earlier studies from other research groups, however, AQP7 was suggested to be able to transport urea as well as glycerol.8,9) The reason for this discrepancy about urea transport is unknown at this time and remains to be resolved in the future. The Km of AQP7 for glycerol (11.9 µM) is comparable with those of AQP9 (9.2 µM) and AQP10 (10.4 µM),6,7) and is relatively close to the glycerol level in the systemic circulation, which could be several tens of µM or greater (up to several hundreds of µM).12) Therefore, it could be assumed that the carrier mode of operation provides a mechanism to restrict the cellular uptake of glycerol, responding to glycerol concentration in plasma. In adipocytes, the major role of AQP7 has been suggested to be its operation for the release of glycerol generated by lipolysis in the cells to plasma.10,11) Since AQP7 has been suggested to be a facilitative carrier, which does not require any major ion for operation, it could also operate for the process of glycerol release. When starved, AQP7 is known to be upregulated in adipocytes to enhance the release of glycerol to increase its supply to the liver, where it is utilized for gluconeogenesis as a mechanism to produce glucose for the survival of the living body.13) More direct evidence for the role of AQP7 in glycerol release in adipocytes has been provided by a study using AQP7-knockout mice,14) in which glycerol was shown to be accumulated more in the knockout mice than in wild-type mice and released more slowly from adipocytes in the former than the latter. Our suggested carrier mode of its operation could be an additional mechanism to adjust glycerol release. Because glycerol concentration is supposed to be greater in adipocytes than in plasma when it is released, AQP7-mediated transport could be almost saturated and, hence, the release rate may be mainly controlled by the maximum transport rate and the number of AQP7 molecules. The characteristics of the carrier mode of AQP7 operation are also quite similar to those of AQP9 and AQP10 in that it is of facilitative type and all the inhibitors are similarly effective. In our previous studies,6,7) AQP9 and AQP10 were found not to require Na+ for their carrier mode of glycerol transport function and, hence, assumed to be facilitative carriers. For AQP10, the pHinsensitive characteristic was also confirmed. In addition, glycerol transports by these AQPs were inhibited extensively by monoacetin, monobutyrin, diglycerol and 1,2-propanediol at their concentration of 10 mM, and, to a lesser extent, by 1,2-ethanediol. Moreover, S-1,2-propanediol was more potent in inhibiting them than R-1,2-propanediol, manifesting the enantioselective characterisitc. Thus, all these characteristics of AQP9 and AQP10 are
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quite similar to those of AQP7 observed in the present study. The only notable difference is that AQP7 is more sensitive to chloramphenicol than AQP9 and AQP10. Whereas AQP7-specific glycerol transport was inhibited by about 85% by this drug (10 mM), the AQP9-specific one was inhibited only modestly by about 30%6) and the AQP10-specific one was not inhibited at all.7) In conclusion, AQP7 was found to function as a facilitative carrier for glycerol and, possibly, also for analogous compounds. This finding redefines the functional characteristics of this protein, which has been implicitly presumed to function as a channel for glycerol as well as for water as a member of AQP water channels, and provides a novel insight into its operation mechanism. This insight will help further explore its potential roles in physiological processes and also in drug disposition.
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