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glucose cotransporter in renal epithelial cells
Life Sciences 71 (2002) 1 – 13 www.elsevier.com/locate/lifescie
Phosphatidylinositol 3-kinase mediates inhibitory effect of angiotensin II on sodium/glucose cotransporter in renal epithelial cells Kazuya Kawano, Akira Ikari *, Mika Nakano, Yasunobu Suketa Department of Environmental Biochemistry and Toxicology, University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yada, Shizuoka city, Shizuoka 422-8526, Japan Received 23 July 2001; accepted 31 October 2001
Abstract Effects of angiotensin II (ANGII) on regulation of sodium/glucose cotransporter (SGLT1) activity were investigated in LLC-PK1 cells, renal proximal epithelial cell line. ANGII inhibited a [14C] methyl-Dglucopyranoside (AMG) uptake into LLC-PK1 cells in a dose-dependent manner. This inhibition was based on a decrease in maximal transport rate (Vmax) of AMG from 2.20 nmol/mg protein/15 min to 1.19 nmol/mg protein/ 15 min, although apparent affinity constant (Km) did not alter. In western blot analysis, protein level of SGLT1 in brush border membrane (BBM) was decreased by ANGII, although total SGLT1 was not altered. In the aspect of intracellular signal transduction, ANGII blocked the formation of cAMP. Pertussis toxin, an inactivator of Gi protein that control intracellular cAMP level, completely prevented the decrease of AMG uptake caused by ANGII. 8-Br-cAMP, a cell membrane permeable cAMP analogue, increased AMG uptake and protein level of SGLT1 in BBM. Both wortmannin and LY294002 that are phosphatidylinositol (PI) 3-kinase inhibitors, inhibited the SGLT1 activity, and also attenuated the effect of 8-Br-cAMP on SGLT1 activity. Those inhibitors prevented the 8-Br-cAMP-induced expression of SGLT1 in plasma membrane. We conclude that ANGII plays an important role in post-translational regulation in SGLT1. Inhibition of SGLT1 translocation is suggested to be caused by inactivation of protein kinase A and decrease of PI 3-kinase activity. D 2002Elsevier Science Inc. All rights reserved. Keywords: Angiotensin II; SGLT1; LLC-PK1 cells; cAMP; PI 3-kinase; Translocation
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Corresponding author. Tel.: +81-54-264-5674; fax: +81-54-264-5672. E-mail address: [email protected] (A. Ikari). 0024-3205/02/$ - see front matter D 2002Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 5 7 3 - 4
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Introduction Glucose absorption in intestine and reabsorption in kidney have important roles in controlling the plasma glucose levels. Especially, glucose reabsorption in kidney is increasing attention as therapeutic targets in patients with diabetes mellitus [1]. Glucose reabsorption in renal proximal tubule is performed by two types of glucose transporter; one is glucose transporter (GLUT) which have facilitated diffusion system [2], the other is sodium/glucose cotransporter (SGLT) which utilized Na+ gradient across the cell membrane [3]. These transporters express in the renal proximal epithelial cells and show a different localization. SGLT localized in the brush border membrane, compared to GLUT in the basolateral membrane [4]. This different localization valuables the transcellular transport of glucose from lumen to serosa. The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure and electrolyte homeostasis. In this system, angiotensin II (ANGII) has potent effect on regulatory mechanism. ANGII has systematically wide and various effects but classically serves to enhance vasoconstriction, aldosterone release, renal sodium transport, renal blood flow, and thirst. RAS is constructed with renin, angiotensinogen, angiotensins, and angiotensin-converting enzyme. All members of RAS had been found in local tissue [5], and activity of local RAS has been concerned in a variety of physiological pathways and pathophysiological conditions [6–9]. In kidney, RAS mainly regulates the electrolyte balance and fluid volume. The mechanism controlling the electrolyte balance is maintained by regulation of Na+ reabsorption in proximal tubule. In this mechanism, although the most important pathway on Na+ reabsorption is Na+/H+ exchanger [10], Na+/H+ exchanger could not account for all Na+ reabsorption. Other Na+-coupled transport processes are suggested to be related to remainder Na+ reabsorption [11]. Na+-dependent glucose and amino acid absorption also must account for net sodium absorption [12,13], but there is no much study that ANGII regulates these Na+-dependent transporters. In general, SGLT1 protein was transported from endoplasmic reticulum to golgi complex and followed by glycosylation. This matured protein is expressed in plasma membrane, and it acts as a functional protein [14]. The post-translational regulation of SGLT1 is divided into two mechanisms; one is the change of affinity for substrate that mediated by direct phosphorylation of amino acid of SGLT1 [15], and the other is regulation of the number of SGLT1 that is expressing in plasma membrane [14]. The latter is explained by the trafficking of cAMP-stimulated SGLT1-containing vesicles from intracellular compartment to plasma membrane. It resulted in the up-regulation of glucose transport activity. In the past reports, several hormones utilize this mechanism to regulate SGLT1 activity. In the intestine of diabetic rat, the abundance of SGLT1 in plasma membrane is restored by insulin [16]. In normal rat intestine, glucagon like peptide-2 (GLP-2) also up-regulated the level of SGLT1 expression in plasma membrane [17]. However, there is little report about hormonal regulation of renal SGLT1. In the present study, we revealed the effect of ANGII on the regulatory mechanism of renal SGLT1 activity.
Methods Materials a [14C] Methyl-D-glucopyranoside (AMG) was purchased from NEN Life Science Products (Boston, MA, U.S.A.). ANGII (Human) was from PEPTIDE INSTITUTE (Osaka, Japan). Wortmannin
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was from Wako Pure Chemical Industries (Osaka, Japan). Phlorizin, 8-Br-cAMP, pertussis toxin (PTX), LY294002 and fetal calf serum were from Sigma (St. Louis, MO, U.S.A.). Trypsin 2.5% was from LIFE TECHNOLOGIES (Tokyo, Japan). Scintizol EX-H was from Dojindo Laboratories (Kumamoto, Japan). HRP-conjugated anti-rabbit IgG whole antibody and ECL Western Blotting Detection Reagents were from Amersham Pharmacia Biotech (Piscatway, U.S.A.). A polyclonal antibody against porcine renal SGLT1 [18] was kindly provided by Prof. Julia E. Lever (University of Texas Medical School, Houston, U.S.A.). Cell Culture The porcine renal cell line, LLC-PK1, obtained from JCRB (Tokyo, Japan) was maintained on plastic dishes (Corning, Tokyo, Japan) in medium 199 supplemented with 10% fetal calf serum under atmosphere of 5% CO2 –95% O2 at 37 jC. Subculture was done every 3–4 days using 0.02% EDTA and 0.25% trypsin. For the transport assay, LLC-PK1 cells were seeded on 24-well culture dishes at a cell density of 1.0 105 cells/cm2 and cultured for 24 h before experiment. Transport Assay Cells were washed two times at 37 jC with Hanks’ balanced salt solution (HBSS; 137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM Hepes, pH 7.4). The transport assay was initiated by the addition of 0.2 ml of HBSS containing 0.5 mM AMG (0.4 ACi/ml) as substrate. The cells were incubated for 15 min at 37 jC, and uptake was terminated by aspiration of the incubation mixture followed immediately by three times rinses with ice-cold HBSS. Then, the cells were solubilized with 0.5 N NaOH and aliquots were taken for determination of radioactivity. We also examined the AMG uptake in the presence of 500 AM phlorizin, a specific inhibitor of SGLT, and this value was subtracted as a non-specific AMG transport and adhesion of cell surface. Protein content of each sample was measured using the method of Bradford, and data were represented as AMG uptake/mg protein/15 min. Cell Membrane Preparation Brush border membrane (BBM) was prepared from LLC-PK1 cells by the procedure of Turner et al. [19], and this BBM fraction was subjected to western blot analysis. Whole cell lysate was prepared by solubilizing with lysis buffer (150 mM NaCl, 0.02% sodium azide, 100 Ag/ml phenylmethylsulfonyl fluoride, 1% Triton X-100 and 50 mM Tris–HCl, pH 8.0) and harvested with a rubber policeman. The lysate was centrifuged at 12,000 g for 2 min at 4 jC. The supernatant was used as whole cell lysate. Western Blot Analysis Samples of BBM and whole cell lysate (20 Ag of total protein) were subjected to SDS-polyacrylamide (10% acrylamide) gel electrophoresis. Proteins and molecular weight markers (Amersham Pharmacia Biotech) were electrophoretically transferred to PVDF membrane (MILLIPORE, Tokyo, Japan) at room temperature using a transfer unit (Bio-Rad Laboratories, CA, U.S.A.). The PVDF membrane was immersed in a blocking solution (0.1% Tween 20, 0.15 M NaCl, 1% bovine serum albumin, and 20 mM Tris–HCl, pH 7.4) for 2 h at room temperature, and incubated with a polyclonal antibody against pig
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renal SGLT1 [18] at 1:2500 dilution for 3 h. The PVDF membrane was washed three times with a washing buffer (0.1% Tween 20, 0.15 M NaCl and 20 mM Tris–HCl, pH 7.4) for 5 min, and incubated in a blocking solution with HRP-conjugated goat anti-rabbit IgG as secondary anti-body at 1:2000 dilution for 3 h. The PVDF membrane was washed three times with a washing buffer for 5 min, and then detection was carried out using ECL Western Blotting Detection Reagents. The PVDF membrane exposed autoradiography film (Hyperfilm ECL, Amersham International) for 1 h, followed by quantification using NIH image 1.61. Statistical Analysis Results are represented as means F S.E. Differences between two groups were made by one-way analysis of variance (ANOVA). Statistical significance is assumed at p < 0.05.
Results Effect of ANGII on SGLT1 Activity in LLC-PK1 Cells LLC-PK1 cells are well-differentiated epithelial cell line derived from porcine renal proximal tubule cells [20], and it is utilized as a model of glucose absorption in renal proximal tubule. We examined the
Fig. 1. Inhibition of SGLT1 activity by ANGII. LLC-PK1 cells, grown in 24-well culture plate, were pre-incubated with indicated concentration of ANGII between 10 16 and 10 6 M for 2 h at 37 jC, followed by incubation with AMG for 15 min. Data are presented as means F S.E. (n = 4). * and **, significantly different from the value in the absence of ANGII ( p < 0.05 and 0.01, respectively).
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.
Fig. 2. Determination of kinetic parameters of AMG uptake. (A). LLC-PK1 cells were pre-incubated in the presence ( ) or absence (n) of ANGII (1 AM) for 2 h at 37 jC. Non-labeled AMG concentrations were varied between 0.05 and 2 mM, and uptake was determined after 15 min incubation. (B). In order to determine the kinetic properties of AMG uptake, the data are presented in a double-reciprocal plot. Data are presented as means F S.E. (n = 3 – 6).
Fig. 3. Increase of SGLT1 activity by 8-Br-cAMP. LLC-PK1 cells were pre-incubated with ANGII (1 AM), 8-Br-cAMP (250 AM), or both of them for 2 h at 37 jC, followed by incubation with AMG for 15 min. Data are presented as means F S.E. (n = 3). *, significantly different from control ( p < 0.05).
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AMG transport properties in LLC-PK1 cells. Pre-treatment of ANGII for 2 h dose-dependently decreased the SGLT1 activity (Fig. 1). The significant effect on AMG uptake emerged more than 1 pM ANGII. Concentration of non-labeled AMG was changed between 0.05 mM and 2 mM, in the presence or absence of ANGII (1 AM) (Fig. 2A). The data were analyzed using a double-reciprocal plot (Fig. 2B). In non-treated cells, an apparent affinity constant (Km) for AMG was 0.39 mM (n = 3–6) and maximal rate of transport (Vmax) was 2.20 nmol/mg protein/15 min. In ANGII treated cells, Km was 0.33 mM (n = 3–6) and Vmax was 1.19 nmol/mg protein/15 min. ANGII potently decreased Vmax values, but did not change Km values for AMG. 8-Br-cAMP Increased SGLT1 Activity and Protein Level in BBM Pre-treatment of 8-Br-cAMP (250 AM) for 2 h resulted in 1.5-fold increase in SGLT1 activity on control cells (Fig. 3). ANGII (1 AM) also inhibited AMG uptake in 8-Br-cAMP treated cells. A specific receptor for ANGII in plasma membrane coupled with Gi protein blocks the formation of intracellular cAMP by binding with ANGII. In determination of quantity of intracellular cAMP, ANGII (1 AM) significantly decreased cAMP content (from: 1.25 F 0.10 pmol/mg protein, to: 0.87 F 0.09 pmol/mg protein (n = 4, p < 0.05)). Although PTX (100 ng/ml), inactivator of Gi protein, prevented the inhibitory effect of ANGII on SGLT1 activity, PTX did not independently affect to SGLT1 activity (Fig. 4). Next, we determined the effect of both ANGII and 8-Br-cAMP on the protein level of SGLT1 in BBM and whole cell lysate. ANGII (1 AM) potently decreased the level of SGLT1 protein in BBM (Fig. 5A). On the contrary, 8-Br-cAMP (250 AM) increased the SGLT1 protein. The stimulating effect of 8-Br-
Fig. 4. Effect of PTX on SGLT1 activity inhibited by ANGII. LLC-PK1 cells were pre-incubated with ANGII (1 AM), PTX (100 ng/ml), or both of them for 2 h at 37 jC, followed by incubation with AMG for 15 min. Data are presented as means F S.E. (n = 4). *, significantly different from control ( p < 0.05). NS, not significantly different (p > 0.05).
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Fig. 5. Protein level of SGLT1 in brush border membrane and whole cell lysate. LLC-PK1 cells were pre-incubated with ANGII (1 AM), 8-Br-cAMP (250 AM), or both of them for 2 h at 37 jC. The BBM fractions (A) and whole cell lysate (B) were prepared as described under methods. Samples (20 Ag/lane) were applied on the gel and blotted with a polyclonal antibody to the pig renal SGLT1. Densitometric values are shown below.
Fig. 6. Inhibition of SGLT1 activity by PI 3-kinase inhibitor. LLC-PK1 cells were pre-incubated with ANGII (1 AM), wortmannin (5 nM) or LY294002 (50 AM) for 2 h at 37 jC, followed by incubation with AMG for 15 min. Data are presented as means F S.E. (n = 3 – 5). **, significantly different from control ( p < 0.01).
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cAMP was suppressed by the treatment of ANGII. In whole cell lysate, the protein level of SGLT1 was not affected by the treatment with ANGII, and 8-Br-cAMP, or both of them (Fig. 5B). PI 3-kinase Inhibitor Decreased SGLT1 Activity and Protein Level PI 3-kinase has an important role in the trafficking of membrane protein from intracellular compartment to plasma membrane [21,22]. In this study, two types of PI 3-kinase inhibitor, wortmannin and LY294002, were used to examine the effect of PI 3-kinase on SGLT1 translocation. Pre-treatment with wortmannin (5 nM) or LY294002 (50 AM) for 2 h inhibited SGLT1 activity (Fig. 6). To investigate the effect of cAMP on PI 3-kinase activity, wortmannin and LY294002 were pre-treated, and then followed by treatment of 8-Br-cAMP (250 AM). Both chemicals inhibited the cAMP-induced activation of AMG uptake (Fig. 7). In western blotting, those inhibitors prevented the 8-Br-cAMP induced expression of SGLT1 in plasma membrane (Fig. 8). This observation coincided with that of SGLT1 activity. Effect of Na+/K +-ATPase and Na+/H + exchanger on SGLT1 Activity SGLT is utilizing Na+ gradient across the cell membrane as a driving force to transport glucose. Intracellular Na+ concentration is affected by the action of Na+/K+-ATPase in basolateral membrane, and Na+/H+ exchanger in brush border membrane. So we examined the effect of ANGII on Na+/K+-ATPase activity. Na+/K+-ATPase activity in control cells was 0.25 F 0.04 Amol Pi/min/mg protein (n = 4), and in ANGII (1 AM) treated cells was 0.26 F 0.01 Amol Pi/min/mg protein (n = 4), so that we could not observe the significant effect of ANGII on Na+/K+-ATPase activity.
Fig. 7. Effect of PI 3-kinase inhibitor in the presence or absence of 8-Br-cAMP on SGLT1 activity. LLC-PK1 cells were preincubated with wortmannin (5 nM) or LY294002 (50 AM) for 30 min at 37 jC, and followed by incubation with 8-Br-cAMP for 2 h. Cells were incubated with AMG for 15 min. Data are presented as means F S.E. (n = 3). * and **, significantly different from the value in control ( p < 0.05 and 0.01, respectively).
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Fig. 8. Protein level of SGLT1 in brush border membrane. LLC-PK1 cells were pre-incubated with 8-Br-cAMP (250 AM) or 8-Br-cAMP and each PI 3-kinase inhibitor (5 nM wortmannin or 50 AM LY294002) for 2 h at 37 jC. The plasma membrane fractions were prepared as described under methods. Samples (20 Ag/lane) were applied on the gel and blotted with a polyclonal antibody to the pig renal SGLT1. Densitometric values are shown below.
It has been reported that ANGII activates Na+/H+ exchanger, which cause intracellular alkalization and result in increasing intracellular Na+ concentration [23–25]. To investigate the effect of Na+/H+ exchanger on SGLT1 activity, Na+/H+ exchanger was forcedly activated by lowering intracellular pH or blocked with 5-(N, N-dimethyl) amiloride (DMA), a specific inhibitor of Na+/H+ exchanger. AMG uptake was 3.8 F 0.2 nmol/mg protein/15 min (n = 3) in control cells and 3.6 F 0.1 nmol/mg protein/ 15 min (n = 3) in alkalinized cells, so that SGLT1 activity was not affected by the change of intracellular pH. Furthermore, AMG uptake was not inhibited by DMA (10 AM) (control cells: 2.69 F 0.13 nmol/mg protein/15 min, DMA-treated cells: 2.16 F 0.22 nmol/mg protein/15 min, n = 3, not significantly different). Therefore, inhibition of AMG uptake caused by ANGII is not modulated via the change of Na+/H+ exchanger activity.
Discussion Urinary Na+ is reabsorbed 70–80% in proximal tubule and this mechanism is controlled by various hormones including ANGII. ANGII reveals a dose–dependent biphasic effect on fluid uptake that is stimulation at low concentrations (1–100 pM) and inhibition at high concentration (0.1–100 AM) [26]. This biphasic effect of ANGII is extremely important to regulate fluid and electrolyte homeostasis, although there is no much evidence to explain this biphasic mechanism. In the results of the past studies,
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ANGII affects transepithelial sodium transport by modulation of Na+/H+ exchanger at luminal membrane [24,25]. At the basolateral membrane, Na+/HCO3 cotransporter [24] and Na+/K+-ATPase [27] are also modulated by ANGII. One- to two-thirds of Na+ absorption in proximal tubule is account for electroneutral process such as Na+/H+ exchanger [10] and up to 25% is electrogenic Na+-dependent glucose and amino acid cotransporters [12,13]. Apical Na+/H+ exchanger is stimulated by ANGII at 10 pM [25], and responsible for the increase of fluid and Na+ absorption. On the inhibition of fluid and Na+ absorption, there is no report about which transporters are concerned with. Recently it has been reported that SGLT also behaves as a water pump [28], so there is much possibility that SGLT activity is altered by ANGII, and may relate to the biphasic mechanism of Na+ absorption. In the present study, we utilized LLC-PK1 cells as a model of glucose absorption in renal proximal tubule. This cell line possesses characteristic of distal portion of proximal tubule [29–32] and expresses two types of SGLTs in BBM. One is SGLT1 that is high affinity glucose transoporter (Km: 0.1– 0.75 mM) [33,34]. Another is SGLT3 that is low affinity one (Km: 6 mM) and once had been referred to as pigSGLT2 [35]. These two SGLTs have different sugar selectivity about transport properties. Glucose transport activity on SGLT1 indicates competitive inhibition by galactose, but SGLT3 does not [33,35]. In this cell line, Na+-dependent glucose transport properties are notably sensitive to galactose [36]. Based on these differences about transport properties of SGLT1 and SGLT3, SGLT1 is recognized as functionally dominant isoform expressing in LLC-PK1 cells. Furthermore, we examined the effect of ANGII on Na+-dependent glucose uptake under the condition of 0.5 mM AMG. This concentration of AMG is close to Km value of SGLT1. Therefore we thought that alteration of glucose transport activity by ANGII was result of inhibition of SGLT1. Our present results indicate that ANGII inhibited AMG uptake in a dose-dependent manner. The apparent Km values for AMG on both control and ANGII pre-treated cells were very close to the high affinity transporter reported in LLC-PK1 cells (0.75 mM) [34]. The Vmax value was decreased by ANGII, although Km was not changed. This result revealed that ANGII did not alter the affinity for AMG but decreased the protein level in plasma membrane. As shown in Fig. 5A, ANGII decreased the protein level of SGLT1 in BBM, but did not change that of whole cell lysate. We suggested that ANGII did not alter the transcriptional level of SGLT1 but changed the proportion of SGLT1 distribution between intracellular pool and plasma membrane. It also can be supported by the change of Vmax by ANGII. These results indicate that decrease in SGLT1 activity by ANGII was caused by lowering the level of SGLT1 expression in plasma membrane. ANGII blocks intracellular cAMP formation by binding to ANGII receptor in plasma membrane which is seven-transmembrane receptor that is coupled with Gi protein. This signaling stimulates Na+/H+ exchanger in brush border membrane, and activates Na+ reabsorption from lumen [23–25]. Wright et al. reported that cAMP regulated the number of SGLT1 in xenopus oocytes plasma membrane [14]. In LLC-PK1 cells, we also observed the phenomenon that 8-Br-cAMP increased SGLT1 activity (Fig. 3) and level of SGLT1 expression in BBM (Fig. 5A). We, therefore, suggest that LLC-PK1 cells have similar signaling pathway of xenopus oocytes to regulate the SGLT1 activity, and alteration of intracellular cAMP level is involved in the inhibition of SGLT1 expression by ANGII. PI 3-kinase has a role in the remodeling of actin skeleton and the vesicle trafficking [37,38]. In the study of vesicle trafficking, GLUT4, one of the Na+-independent glucose transporters, is transported from cytosolic compartments to plasma membrane by insulin stimulation [38–40]. Moreover, vesicle trafficking of some Na+-dependent transporters except for SGLT is regulated by the cAMP-mediated PI 3-kinase signaling [21,22]. To make clear the involvement of PI 3-kinase in SGLT1 translocation, we
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used two types of specific PI 3-kinase inhibitors, wortmannin and LY294002. These PI 3-kinase inhibitors are chemically unrelated each other and impair the enzyme by different mechanisms. Wortmannin rapidly and irreversibly inhibits the PI 3-kinase by alkylating the catalytic p110 subunit with an IC50 of 5–10 nM [41]. LY294002 interacts with the ATP-binding site of the enzyme, and inhibits PI 3-kinase with an IC50 within micromolar range [42]. In this study, both wortmannin and LY294002 decreased AMG uptake as well as decrease by ANGII. This result convinced us that PI 3-kinase has an essential role on translocation and activation of SGLT1. This role of PI 3-kinase is also suggested to be involved in cAMP-mediated translocation of SGLT1. This aspect is supported by our finding that PI 3-kinase inhibitors prevents the cAMP-induced activation of AMG uptake (Fig. 7) and expression of SGLT1 in plasma membrane (Fig. 8) in the presence of PI 3-kinase inhibitors. SGLT is utilizing Na+ gradient across the cell membrane as a driving force to transport glucose. Intracellular Na+ concentration is affected by Na+/K+-ATPase in basolateral membrane, and Na+/H+ exchanger in brush border membrane. We investigated the effect of ANGII on Na+/K+-ATPase activity. Na+/K+-ATPase activity was not significantly altered by the treatment of 1 AM ANGII in comparison with control cells. It seems that this inhibitory mechanism was not due to the change of Na+/K+-ATPase activity. Next, we examined the influence of Na+/H+ exchanger upon SGLT1 activity. Na+/H+ exchanger was activated by intracellular acid load in many cells [23–25]. We, however, could not observe that SGLT1 activity was changed by intracellular acid load. Thus, we confirmed that ANGII had direct influence to SGLT1, and the change of intracellular pH and Na+ influx via Na+/H+ exchanger were unrelated to SGLT1 activity. In conclusion, we found that ANGII inhibited SGLT1 activity by blocking intracellular cAMP formation and the translocation of vesicles containing SGLT1 protein. In general, ANGII stimulates Na+ reabsorption in proximal tubule, although our present study demonstrated that Na+ and glucose transports through SGLT1 were decreased by ANGII. This result may explain the biphasic mechanism of ANGII on Na+ reabsorption. Additional studies in terms of ANGII on SGLT1 activity may be helpful in ultimately determining the significance in the physiological and pathological aspects. Acknowledgements We thank Prof. Julia E. Lever of University of Texas Medical School (Houston, U.S.A.) for providing a polyclonal antibody for porcine renal SGLT1.
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[32] Rabito CA, Karish MV. Polarized amino acid transport by an epithelial cell line of renal origin (LLC-PK1). The apical systems. J Biol Chem 1983;258(4):2543 – 7. [33] Panayotova-Heiermann M, Loo DD, Wright EM. Kinetics of steady-state currents and charge movements associated with the rat Na+/glucose cotransporter. J Biol Chem 1995;270(45):27099 – 105. [34] Rabito CA, Ausiello DA. Na+-dependent sugar transport in a cultured epithelial cell line from pig kidney. J Membr Biol 1980;54(1):31 – 8. [35] Mackenzie B, Loo DD, Panayotova-Heiermann M, Wright EM. Biophysical characteristics of the pig kidney Na+/glucose cotransporter SGLT2 reveal a common mechanism for SGLT1 and SGLT2. J Biol Chem 1996;271(51):32678 – 83. [36] Moran A, Handler JS, Turner RJ. Na+-dependent hexose transport in vesicles from cultured renal epithelial cell line. Am J Physiol 1982;243(5):C293 – 8. [37] Khayat ZA, Tong P, Yaworsky K, Bloch RJ, Klip A. Insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol 3-kinase and GLUT4 in L6 myotubes. J Cell Sci 2000;113(Pt 2):279 – 90. [38] White MF, Kahn CR. The insulin signaling system. J Biol Chem 1994;269(1):1 – 4. [39] Braiman L, Sheffi-Friedman L, Bak A, Tennenbaum T, Sampson SR. Tyrosine phosphorylation of specific protein kinase C isoenzymes participates in insulin stimulation of glucose transport in primary cultures of rat skeletal muscle. Diabetes 1999;48(10):1922 – 9. [40] James DE, Strube M, Mueckler M. Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature 1989;338(6210):83 – 7. [41] Arcaro A, Wymann MP. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem J 1993;296(Pt 2):297 – 301. [42] Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 1994;269(7):5241 – 8.
Phosphatidylinositol 3-kinase mediates inhibitory effect of angiotensin II on sodium/glucose cotransporter in renal epithelial cells Kazuya Kawano, Akira Ikari *, Mika Nakano, Yasunobu Suketa Department of Environmental Biochemistry and Toxicology, University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yada, Shizuoka city, Shizuoka 422-8526, Japan Received 23 July 2001; accepted 31 October 2001
Abstract Effects of angiotensin II (ANGII) on regulation of sodium/glucose cotransporter (SGLT1) activity were investigated in LLC-PK1 cells, renal proximal epithelial cell line. ANGII inhibited a [14C] methyl-Dglucopyranoside (AMG) uptake into LLC-PK1 cells in a dose-dependent manner. This inhibition was based on a decrease in maximal transport rate (Vmax) of AMG from 2.20 nmol/mg protein/15 min to 1.19 nmol/mg protein/ 15 min, although apparent affinity constant (Km) did not alter. In western blot analysis, protein level of SGLT1 in brush border membrane (BBM) was decreased by ANGII, although total SGLT1 was not altered. In the aspect of intracellular signal transduction, ANGII blocked the formation of cAMP. Pertussis toxin, an inactivator of Gi protein that control intracellular cAMP level, completely prevented the decrease of AMG uptake caused by ANGII. 8-Br-cAMP, a cell membrane permeable cAMP analogue, increased AMG uptake and protein level of SGLT1 in BBM. Both wortmannin and LY294002 that are phosphatidylinositol (PI) 3-kinase inhibitors, inhibited the SGLT1 activity, and also attenuated the effect of 8-Br-cAMP on SGLT1 activity. Those inhibitors prevented the 8-Br-cAMP-induced expression of SGLT1 in plasma membrane. We conclude that ANGII plays an important role in post-translational regulation in SGLT1. Inhibition of SGLT1 translocation is suggested to be caused by inactivation of protein kinase A and decrease of PI 3-kinase activity. D 2002Elsevier Science Inc. All rights reserved. Keywords: Angiotensin II; SGLT1; LLC-PK1 cells; cAMP; PI 3-kinase; Translocation
*
Corresponding author. Tel.: +81-54-264-5674; fax: +81-54-264-5672. E-mail address: [email protected] (A. Ikari). 0024-3205/02/$ - see front matter D 2002Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 5 7 3 - 4
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Introduction Glucose absorption in intestine and reabsorption in kidney have important roles in controlling the plasma glucose levels. Especially, glucose reabsorption in kidney is increasing attention as therapeutic targets in patients with diabetes mellitus [1]. Glucose reabsorption in renal proximal tubule is performed by two types of glucose transporter; one is glucose transporter (GLUT) which have facilitated diffusion system [2], the other is sodium/glucose cotransporter (SGLT) which utilized Na+ gradient across the cell membrane [3]. These transporters express in the renal proximal epithelial cells and show a different localization. SGLT localized in the brush border membrane, compared to GLUT in the basolateral membrane [4]. This different localization valuables the transcellular transport of glucose from lumen to serosa. The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure and electrolyte homeostasis. In this system, angiotensin II (ANGII) has potent effect on regulatory mechanism. ANGII has systematically wide and various effects but classically serves to enhance vasoconstriction, aldosterone release, renal sodium transport, renal blood flow, and thirst. RAS is constructed with renin, angiotensinogen, angiotensins, and angiotensin-converting enzyme. All members of RAS had been found in local tissue [5], and activity of local RAS has been concerned in a variety of physiological pathways and pathophysiological conditions [6–9]. In kidney, RAS mainly regulates the electrolyte balance and fluid volume. The mechanism controlling the electrolyte balance is maintained by regulation of Na+ reabsorption in proximal tubule. In this mechanism, although the most important pathway on Na+ reabsorption is Na+/H+ exchanger [10], Na+/H+ exchanger could not account for all Na+ reabsorption. Other Na+-coupled transport processes are suggested to be related to remainder Na+ reabsorption [11]. Na+-dependent glucose and amino acid absorption also must account for net sodium absorption [12,13], but there is no much study that ANGII regulates these Na+-dependent transporters. In general, SGLT1 protein was transported from endoplasmic reticulum to golgi complex and followed by glycosylation. This matured protein is expressed in plasma membrane, and it acts as a functional protein [14]. The post-translational regulation of SGLT1 is divided into two mechanisms; one is the change of affinity for substrate that mediated by direct phosphorylation of amino acid of SGLT1 [15], and the other is regulation of the number of SGLT1 that is expressing in plasma membrane [14]. The latter is explained by the trafficking of cAMP-stimulated SGLT1-containing vesicles from intracellular compartment to plasma membrane. It resulted in the up-regulation of glucose transport activity. In the past reports, several hormones utilize this mechanism to regulate SGLT1 activity. In the intestine of diabetic rat, the abundance of SGLT1 in plasma membrane is restored by insulin [16]. In normal rat intestine, glucagon like peptide-2 (GLP-2) also up-regulated the level of SGLT1 expression in plasma membrane [17]. However, there is little report about hormonal regulation of renal SGLT1. In the present study, we revealed the effect of ANGII on the regulatory mechanism of renal SGLT1 activity.
Methods Materials a [14C] Methyl-D-glucopyranoside (AMG) was purchased from NEN Life Science Products (Boston, MA, U.S.A.). ANGII (Human) was from PEPTIDE INSTITUTE (Osaka, Japan). Wortmannin
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was from Wako Pure Chemical Industries (Osaka, Japan). Phlorizin, 8-Br-cAMP, pertussis toxin (PTX), LY294002 and fetal calf serum were from Sigma (St. Louis, MO, U.S.A.). Trypsin 2.5% was from LIFE TECHNOLOGIES (Tokyo, Japan). Scintizol EX-H was from Dojindo Laboratories (Kumamoto, Japan). HRP-conjugated anti-rabbit IgG whole antibody and ECL Western Blotting Detection Reagents were from Amersham Pharmacia Biotech (Piscatway, U.S.A.). A polyclonal antibody against porcine renal SGLT1 [18] was kindly provided by Prof. Julia E. Lever (University of Texas Medical School, Houston, U.S.A.). Cell Culture The porcine renal cell line, LLC-PK1, obtained from JCRB (Tokyo, Japan) was maintained on plastic dishes (Corning, Tokyo, Japan) in medium 199 supplemented with 10% fetal calf serum under atmosphere of 5% CO2 –95% O2 at 37 jC. Subculture was done every 3–4 days using 0.02% EDTA and 0.25% trypsin. For the transport assay, LLC-PK1 cells were seeded on 24-well culture dishes at a cell density of 1.0 105 cells/cm2 and cultured for 24 h before experiment. Transport Assay Cells were washed two times at 37 jC with Hanks’ balanced salt solution (HBSS; 137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM Hepes, pH 7.4). The transport assay was initiated by the addition of 0.2 ml of HBSS containing 0.5 mM AMG (0.4 ACi/ml) as substrate. The cells were incubated for 15 min at 37 jC, and uptake was terminated by aspiration of the incubation mixture followed immediately by three times rinses with ice-cold HBSS. Then, the cells were solubilized with 0.5 N NaOH and aliquots were taken for determination of radioactivity. We also examined the AMG uptake in the presence of 500 AM phlorizin, a specific inhibitor of SGLT, and this value was subtracted as a non-specific AMG transport and adhesion of cell surface. Protein content of each sample was measured using the method of Bradford, and data were represented as AMG uptake/mg protein/15 min. Cell Membrane Preparation Brush border membrane (BBM) was prepared from LLC-PK1 cells by the procedure of Turner et al. [19], and this BBM fraction was subjected to western blot analysis. Whole cell lysate was prepared by solubilizing with lysis buffer (150 mM NaCl, 0.02% sodium azide, 100 Ag/ml phenylmethylsulfonyl fluoride, 1% Triton X-100 and 50 mM Tris–HCl, pH 8.0) and harvested with a rubber policeman. The lysate was centrifuged at 12,000 g for 2 min at 4 jC. The supernatant was used as whole cell lysate. Western Blot Analysis Samples of BBM and whole cell lysate (20 Ag of total protein) were subjected to SDS-polyacrylamide (10% acrylamide) gel electrophoresis. Proteins and molecular weight markers (Amersham Pharmacia Biotech) were electrophoretically transferred to PVDF membrane (MILLIPORE, Tokyo, Japan) at room temperature using a transfer unit (Bio-Rad Laboratories, CA, U.S.A.). The PVDF membrane was immersed in a blocking solution (0.1% Tween 20, 0.15 M NaCl, 1% bovine serum albumin, and 20 mM Tris–HCl, pH 7.4) for 2 h at room temperature, and incubated with a polyclonal antibody against pig
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renal SGLT1 [18] at 1:2500 dilution for 3 h. The PVDF membrane was washed three times with a washing buffer (0.1% Tween 20, 0.15 M NaCl and 20 mM Tris–HCl, pH 7.4) for 5 min, and incubated in a blocking solution with HRP-conjugated goat anti-rabbit IgG as secondary anti-body at 1:2000 dilution for 3 h. The PVDF membrane was washed three times with a washing buffer for 5 min, and then detection was carried out using ECL Western Blotting Detection Reagents. The PVDF membrane exposed autoradiography film (Hyperfilm ECL, Amersham International) for 1 h, followed by quantification using NIH image 1.61. Statistical Analysis Results are represented as means F S.E. Differences between two groups were made by one-way analysis of variance (ANOVA). Statistical significance is assumed at p < 0.05.
Results Effect of ANGII on SGLT1 Activity in LLC-PK1 Cells LLC-PK1 cells are well-differentiated epithelial cell line derived from porcine renal proximal tubule cells [20], and it is utilized as a model of glucose absorption in renal proximal tubule. We examined the
Fig. 1. Inhibition of SGLT1 activity by ANGII. LLC-PK1 cells, grown in 24-well culture plate, were pre-incubated with indicated concentration of ANGII between 10 16 and 10 6 M for 2 h at 37 jC, followed by incubation with AMG for 15 min. Data are presented as means F S.E. (n = 4). * and **, significantly different from the value in the absence of ANGII ( p < 0.05 and 0.01, respectively).
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Fig. 2. Determination of kinetic parameters of AMG uptake. (A). LLC-PK1 cells were pre-incubated in the presence ( ) or absence (n) of ANGII (1 AM) for 2 h at 37 jC. Non-labeled AMG concentrations were varied between 0.05 and 2 mM, and uptake was determined after 15 min incubation. (B). In order to determine the kinetic properties of AMG uptake, the data are presented in a double-reciprocal plot. Data are presented as means F S.E. (n = 3 – 6).
Fig. 3. Increase of SGLT1 activity by 8-Br-cAMP. LLC-PK1 cells were pre-incubated with ANGII (1 AM), 8-Br-cAMP (250 AM), or both of them for 2 h at 37 jC, followed by incubation with AMG for 15 min. Data are presented as means F S.E. (n = 3). *, significantly different from control ( p < 0.05).
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AMG transport properties in LLC-PK1 cells. Pre-treatment of ANGII for 2 h dose-dependently decreased the SGLT1 activity (Fig. 1). The significant effect on AMG uptake emerged more than 1 pM ANGII. Concentration of non-labeled AMG was changed between 0.05 mM and 2 mM, in the presence or absence of ANGII (1 AM) (Fig. 2A). The data were analyzed using a double-reciprocal plot (Fig. 2B). In non-treated cells, an apparent affinity constant (Km) for AMG was 0.39 mM (n = 3–6) and maximal rate of transport (Vmax) was 2.20 nmol/mg protein/15 min. In ANGII treated cells, Km was 0.33 mM (n = 3–6) and Vmax was 1.19 nmol/mg protein/15 min. ANGII potently decreased Vmax values, but did not change Km values for AMG. 8-Br-cAMP Increased SGLT1 Activity and Protein Level in BBM Pre-treatment of 8-Br-cAMP (250 AM) for 2 h resulted in 1.5-fold increase in SGLT1 activity on control cells (Fig. 3). ANGII (1 AM) also inhibited AMG uptake in 8-Br-cAMP treated cells. A specific receptor for ANGII in plasma membrane coupled with Gi protein blocks the formation of intracellular cAMP by binding with ANGII. In determination of quantity of intracellular cAMP, ANGII (1 AM) significantly decreased cAMP content (from: 1.25 F 0.10 pmol/mg protein, to: 0.87 F 0.09 pmol/mg protein (n = 4, p < 0.05)). Although PTX (100 ng/ml), inactivator of Gi protein, prevented the inhibitory effect of ANGII on SGLT1 activity, PTX did not independently affect to SGLT1 activity (Fig. 4). Next, we determined the effect of both ANGII and 8-Br-cAMP on the protein level of SGLT1 in BBM and whole cell lysate. ANGII (1 AM) potently decreased the level of SGLT1 protein in BBM (Fig. 5A). On the contrary, 8-Br-cAMP (250 AM) increased the SGLT1 protein. The stimulating effect of 8-Br-
Fig. 4. Effect of PTX on SGLT1 activity inhibited by ANGII. LLC-PK1 cells were pre-incubated with ANGII (1 AM), PTX (100 ng/ml), or both of them for 2 h at 37 jC, followed by incubation with AMG for 15 min. Data are presented as means F S.E. (n = 4). *, significantly different from control ( p < 0.05). NS, not significantly different (p > 0.05).
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Fig. 5. Protein level of SGLT1 in brush border membrane and whole cell lysate. LLC-PK1 cells were pre-incubated with ANGII (1 AM), 8-Br-cAMP (250 AM), or both of them for 2 h at 37 jC. The BBM fractions (A) and whole cell lysate (B) were prepared as described under methods. Samples (20 Ag/lane) were applied on the gel and blotted with a polyclonal antibody to the pig renal SGLT1. Densitometric values are shown below.
Fig. 6. Inhibition of SGLT1 activity by PI 3-kinase inhibitor. LLC-PK1 cells were pre-incubated with ANGII (1 AM), wortmannin (5 nM) or LY294002 (50 AM) for 2 h at 37 jC, followed by incubation with AMG for 15 min. Data are presented as means F S.E. (n = 3 – 5). **, significantly different from control ( p < 0.01).
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cAMP was suppressed by the treatment of ANGII. In whole cell lysate, the protein level of SGLT1 was not affected by the treatment with ANGII, and 8-Br-cAMP, or both of them (Fig. 5B). PI 3-kinase Inhibitor Decreased SGLT1 Activity and Protein Level PI 3-kinase has an important role in the trafficking of membrane protein from intracellular compartment to plasma membrane [21,22]. In this study, two types of PI 3-kinase inhibitor, wortmannin and LY294002, were used to examine the effect of PI 3-kinase on SGLT1 translocation. Pre-treatment with wortmannin (5 nM) or LY294002 (50 AM) for 2 h inhibited SGLT1 activity (Fig. 6). To investigate the effect of cAMP on PI 3-kinase activity, wortmannin and LY294002 were pre-treated, and then followed by treatment of 8-Br-cAMP (250 AM). Both chemicals inhibited the cAMP-induced activation of AMG uptake (Fig. 7). In western blotting, those inhibitors prevented the 8-Br-cAMP induced expression of SGLT1 in plasma membrane (Fig. 8). This observation coincided with that of SGLT1 activity. Effect of Na+/K +-ATPase and Na+/H + exchanger on SGLT1 Activity SGLT is utilizing Na+ gradient across the cell membrane as a driving force to transport glucose. Intracellular Na+ concentration is affected by the action of Na+/K+-ATPase in basolateral membrane, and Na+/H+ exchanger in brush border membrane. So we examined the effect of ANGII on Na+/K+-ATPase activity. Na+/K+-ATPase activity in control cells was 0.25 F 0.04 Amol Pi/min/mg protein (n = 4), and in ANGII (1 AM) treated cells was 0.26 F 0.01 Amol Pi/min/mg protein (n = 4), so that we could not observe the significant effect of ANGII on Na+/K+-ATPase activity.
Fig. 7. Effect of PI 3-kinase inhibitor in the presence or absence of 8-Br-cAMP on SGLT1 activity. LLC-PK1 cells were preincubated with wortmannin (5 nM) or LY294002 (50 AM) for 30 min at 37 jC, and followed by incubation with 8-Br-cAMP for 2 h. Cells were incubated with AMG for 15 min. Data are presented as means F S.E. (n = 3). * and **, significantly different from the value in control ( p < 0.05 and 0.01, respectively).
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Fig. 8. Protein level of SGLT1 in brush border membrane. LLC-PK1 cells were pre-incubated with 8-Br-cAMP (250 AM) or 8-Br-cAMP and each PI 3-kinase inhibitor (5 nM wortmannin or 50 AM LY294002) for 2 h at 37 jC. The plasma membrane fractions were prepared as described under methods. Samples (20 Ag/lane) were applied on the gel and blotted with a polyclonal antibody to the pig renal SGLT1. Densitometric values are shown below.
It has been reported that ANGII activates Na+/H+ exchanger, which cause intracellular alkalization and result in increasing intracellular Na+ concentration [23–25]. To investigate the effect of Na+/H+ exchanger on SGLT1 activity, Na+/H+ exchanger was forcedly activated by lowering intracellular pH or blocked with 5-(N, N-dimethyl) amiloride (DMA), a specific inhibitor of Na+/H+ exchanger. AMG uptake was 3.8 F 0.2 nmol/mg protein/15 min (n = 3) in control cells and 3.6 F 0.1 nmol/mg protein/ 15 min (n = 3) in alkalinized cells, so that SGLT1 activity was not affected by the change of intracellular pH. Furthermore, AMG uptake was not inhibited by DMA (10 AM) (control cells: 2.69 F 0.13 nmol/mg protein/15 min, DMA-treated cells: 2.16 F 0.22 nmol/mg protein/15 min, n = 3, not significantly different). Therefore, inhibition of AMG uptake caused by ANGII is not modulated via the change of Na+/H+ exchanger activity.
Discussion Urinary Na+ is reabsorbed 70–80% in proximal tubule and this mechanism is controlled by various hormones including ANGII. ANGII reveals a dose–dependent biphasic effect on fluid uptake that is stimulation at low concentrations (1–100 pM) and inhibition at high concentration (0.1–100 AM) [26]. This biphasic effect of ANGII is extremely important to regulate fluid and electrolyte homeostasis, although there is no much evidence to explain this biphasic mechanism. In the results of the past studies,
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ANGII affects transepithelial sodium transport by modulation of Na+/H+ exchanger at luminal membrane [24,25]. At the basolateral membrane, Na+/HCO3 cotransporter [24] and Na+/K+-ATPase [27] are also modulated by ANGII. One- to two-thirds of Na+ absorption in proximal tubule is account for electroneutral process such as Na+/H+ exchanger [10] and up to 25% is electrogenic Na+-dependent glucose and amino acid cotransporters [12,13]. Apical Na+/H+ exchanger is stimulated by ANGII at 10 pM [25], and responsible for the increase of fluid and Na+ absorption. On the inhibition of fluid and Na+ absorption, there is no report about which transporters are concerned with. Recently it has been reported that SGLT also behaves as a water pump [28], so there is much possibility that SGLT activity is altered by ANGII, and may relate to the biphasic mechanism of Na+ absorption. In the present study, we utilized LLC-PK1 cells as a model of glucose absorption in renal proximal tubule. This cell line possesses characteristic of distal portion of proximal tubule [29–32] and expresses two types of SGLTs in BBM. One is SGLT1 that is high affinity glucose transoporter (Km: 0.1– 0.75 mM) [33,34]. Another is SGLT3 that is low affinity one (Km: 6 mM) and once had been referred to as pigSGLT2 [35]. These two SGLTs have different sugar selectivity about transport properties. Glucose transport activity on SGLT1 indicates competitive inhibition by galactose, but SGLT3 does not [33,35]. In this cell line, Na+-dependent glucose transport properties are notably sensitive to galactose [36]. Based on these differences about transport properties of SGLT1 and SGLT3, SGLT1 is recognized as functionally dominant isoform expressing in LLC-PK1 cells. Furthermore, we examined the effect of ANGII on Na+-dependent glucose uptake under the condition of 0.5 mM AMG. This concentration of AMG is close to Km value of SGLT1. Therefore we thought that alteration of glucose transport activity by ANGII was result of inhibition of SGLT1. Our present results indicate that ANGII inhibited AMG uptake in a dose-dependent manner. The apparent Km values for AMG on both control and ANGII pre-treated cells were very close to the high affinity transporter reported in LLC-PK1 cells (0.75 mM) [34]. The Vmax value was decreased by ANGII, although Km was not changed. This result revealed that ANGII did not alter the affinity for AMG but decreased the protein level in plasma membrane. As shown in Fig. 5A, ANGII decreased the protein level of SGLT1 in BBM, but did not change that of whole cell lysate. We suggested that ANGII did not alter the transcriptional level of SGLT1 but changed the proportion of SGLT1 distribution between intracellular pool and plasma membrane. It also can be supported by the change of Vmax by ANGII. These results indicate that decrease in SGLT1 activity by ANGII was caused by lowering the level of SGLT1 expression in plasma membrane. ANGII blocks intracellular cAMP formation by binding to ANGII receptor in plasma membrane which is seven-transmembrane receptor that is coupled with Gi protein. This signaling stimulates Na+/H+ exchanger in brush border membrane, and activates Na+ reabsorption from lumen [23–25]. Wright et al. reported that cAMP regulated the number of SGLT1 in xenopus oocytes plasma membrane [14]. In LLC-PK1 cells, we also observed the phenomenon that 8-Br-cAMP increased SGLT1 activity (Fig. 3) and level of SGLT1 expression in BBM (Fig. 5A). We, therefore, suggest that LLC-PK1 cells have similar signaling pathway of xenopus oocytes to regulate the SGLT1 activity, and alteration of intracellular cAMP level is involved in the inhibition of SGLT1 expression by ANGII. PI 3-kinase has a role in the remodeling of actin skeleton and the vesicle trafficking [37,38]. In the study of vesicle trafficking, GLUT4, one of the Na+-independent glucose transporters, is transported from cytosolic compartments to plasma membrane by insulin stimulation [38–40]. Moreover, vesicle trafficking of some Na+-dependent transporters except for SGLT is regulated by the cAMP-mediated PI 3-kinase signaling [21,22]. To make clear the involvement of PI 3-kinase in SGLT1 translocation, we
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used two types of specific PI 3-kinase inhibitors, wortmannin and LY294002. These PI 3-kinase inhibitors are chemically unrelated each other and impair the enzyme by different mechanisms. Wortmannin rapidly and irreversibly inhibits the PI 3-kinase by alkylating the catalytic p110 subunit with an IC50 of 5–10 nM [41]. LY294002 interacts with the ATP-binding site of the enzyme, and inhibits PI 3-kinase with an IC50 within micromolar range [42]. In this study, both wortmannin and LY294002 decreased AMG uptake as well as decrease by ANGII. This result convinced us that PI 3-kinase has an essential role on translocation and activation of SGLT1. This role of PI 3-kinase is also suggested to be involved in cAMP-mediated translocation of SGLT1. This aspect is supported by our finding that PI 3-kinase inhibitors prevents the cAMP-induced activation of AMG uptake (Fig. 7) and expression of SGLT1 in plasma membrane (Fig. 8) in the presence of PI 3-kinase inhibitors. SGLT is utilizing Na+ gradient across the cell membrane as a driving force to transport glucose. Intracellular Na+ concentration is affected by Na+/K+-ATPase in basolateral membrane, and Na+/H+ exchanger in brush border membrane. We investigated the effect of ANGII on Na+/K+-ATPase activity. Na+/K+-ATPase activity was not significantly altered by the treatment of 1 AM ANGII in comparison with control cells. It seems that this inhibitory mechanism was not due to the change of Na+/K+-ATPase activity. Next, we examined the influence of Na+/H+ exchanger upon SGLT1 activity. Na+/H+ exchanger was activated by intracellular acid load in many cells [23–25]. We, however, could not observe that SGLT1 activity was changed by intracellular acid load. Thus, we confirmed that ANGII had direct influence to SGLT1, and the change of intracellular pH and Na+ influx via Na+/H+ exchanger were unrelated to SGLT1 activity. In conclusion, we found that ANGII inhibited SGLT1 activity by blocking intracellular cAMP formation and the translocation of vesicles containing SGLT1 protein. In general, ANGII stimulates Na+ reabsorption in proximal tubule, although our present study demonstrated that Na+ and glucose transports through SGLT1 were decreased by ANGII. This result may explain the biphasic mechanism of ANGII on Na+ reabsorption. Additional studies in terms of ANGII on SGLT1 activity may be helpful in ultimately determining the significance in the physiological and pathological aspects. Acknowledgements We thank Prof. Julia E. Lever of University of Texas Medical School (Houston, U.S.A.) for providing a polyclonal antibody for porcine renal SGLT1.
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