Protein Kinase B/Akt Mediates cAMP- and Cell Swelling-stimulated Na+/Taurocholate Cotransport and Ntcp Translocation*

Cyclic AMP and cell swelling stimulate hepatic Na+/TC cotransport and Ntcp translocation via the phosphoinositide 3-kinase signaling pathway. To determine the downstream target of the phosphoinositide 3-kinase action, we examined the role of protein kinase B (PKB)/Akt using SB203580 in hepatocytes as well as by transfection with a dominant negative (DN-PKB) or a constitutively active (CA-PKB) form of PKB in HuH-Ntcp cells. Both cAMP and cell swelling stimulated p38 mitogen-activated protein (MAP) kinase as well as PKB activity. Although 100 μm SB203580 inhibited cell swelling- and 8-chlorophenylthio-cAMP-induced activation of both p38 MAP kinase and PKB, 1 μm SB203580 inhibited activation of p38 MAP kinase, but not of PKB, in hepatocytes. 100 μm, but not 1 μm SB203580, inhibited cell swelling- and cAMP-induced increases in taurocholate (TC) uptake and Ntcp translocation in hepatocytes. TC uptake in HuH-Ntcp cells was more than 90% dependent on extracellular Na+. Cyclic AMP and cell swelling increased TC uptake by 50–100% and PKB activity 2–4-fold in HuH-Ntcp cells transfected with the empty vector and failed to increase PKB activity, TC uptake, and Ntcp translocation in DN-PKB-transfected HuH-Ntcp cells. Transfection with CA-PKB increased PKB activity, TC uptake, and Ntcp translocation in HuH-Ntcp cells compared with cells transfected with the empty vector. In contrast, transfection with DN-PKB did not affect basal PKB activity, TC uptake, or Ntcp translocation. Taken together, these results strongly suggest that cell swelling and cAMP-mediated stimulation of hepatic Na+/TC cotransport and Ntcp translocation requires activation of PKB and is mediated at least in part via a phosphoinositide 3-kinase/PKB-signaling pathway.

It is now well recognized that biological effects of various hormones and growth factors are mediated via the PI 3-kinase 1 signaling pathway (1,2). Two classes of serine/threonine kinases, namely PKB (also known as Akt) and PKC /, have been proposed to act downstream of PI 3-kinase (2). PKB requires sequential phosphorylation to Thr 308 and Ser 473 by phosphoinositide-dependent protein kinase 1 (PDK1) and PDK2, respectively, to be fully active (3). PKB has been suggested to mediate the transduction of cell survival signals and glucose metabolism (1,2). More specifically, PKB has been shown to protect hepatocytes from tumor necrosis factor ␣and Fas-mediated apoptosis (4) and to regulate translocation of GLUT4 to the plasma membrane (5)(6)(7)(8) and inter-endosomal GLUT4 trafficking (9). Although PI 3-kinase has been shown to be involved in the regulation of a number of hepatobiliary transporters (10 -13), the role of PKB in the regulation of these transporters has not been established.
Conjugated bile acids like TC are efficiently taken up by hepatocytes primarily via a Na ϩ /TC cotransport mechanism, and Ntcp, a serine/threonine phosphoprotein (14), is considered to be the major protein involved in Na ϩ /TC cotransport (15)(16)(17). Ntcp mRNA and Na ϩ /TC cotransport have been shown to be down-regulated in experimental cholestasis and up-regulated by prolactin (18 -22), indicating transcriptional/translational regulation of Ntcp. In addition, Ntcp undergoes posttranslational regulation as indicated by the results which show that cAMP and cell swelling rapidly increase Na ϩ /TC cotransport and Ntcp translocation to the plasma membrane (23,24). Cell swelling and cAMP activate PKB in hepatocytes, and wortmannin, an inhibitor of PI 3-kinase, inhibits the increases in PKB activity and Ntcp translocation induced by cAMP and cell swelling (23,24). These results raise the possibility that PKB may be involved in the regulation of Na ϩ /TC cotransport and Ntcp translocation in hepatocytes.
The aim of the present study was to determine whether the effect of cAMP and cell swelling on Na ϩ /TC cotransport and Ntcp translocation is mediated via PKB. The role of PKB was evaluated by SB203580, a known inhibitor of p38 MAP kinase, which has recently been shown to inhibit PKB at higher concentrations in human and murine T cells (25). In addition, we studied the effect of transient transfection with a dominant negative or a constitutively active PKB construct on TC uptake in HuH-Ntcp cell lines. Results suggest that activation of PKB is necessary for cell swelling-and cAMP-mediated increases in Na ϩ /TC cotransport and Ntcp translocation.
Hepatocytes Preparation-Hepatocytes were isolated from rat livers using a previously described collagenase perfusion method (30). Freshly prepared hepatocytes suspended (100 mg of wet weight/ml) in a HEPES assay buffer (pH 7.4) containing 20 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgSO 4 , 1.0 mM CaCl 2 , 0.8 mM KH 2 PO 4 , and 5 mM glucose were incubated for 30 min at 37°C under air before initiating studies. Hepatocytes were pretreated with SB203580 (1-100 M) for 30 min followed by incubation with 10 M CPT-cAMP for 15 min or hypotonic solution (160 mosmol) for 25 min as previously described (23,24). This was followed by determination of TC uptake, Ntcp translocation, and activities of p38 MAP kinase and PKB. Details of these experiments are given in the legends of each figure. All studies were repeated in at least three different cell preparations.
HuH-Ntcp Cell Culture and Transfections-HuH-Ntcp cells were cultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 100,000 units/liter penicillin, 100 mg/liter streptomycin, 1.2 g/liter G418 at 37°C in a 5% CO 2 , 95% O 2 air incubator. For transfection experiments, cells (3 ϫ 10 5 cells/well in 12-well plates) were cultured for 24 h in the culture medium. Cells were then transiently transfected with a PKB plasmid using LipofectAMINE ac-cording to the instructions provided by the manufacturer. Briefly, cells were transfected by adding 0.5 ml of Opti-MEM I containing 0.0025 l LipofectAMINE and 0.5 g of dominant negative (DN)-PKB or CA-PKB. After 24 h of incubation in the transfection medium, cells were cultured for an additional 24 h in 0.5 ml of culture media. Cells were then incubated in HEPES buffer (above) for 3 h at 37°C before determining TC uptake, Ntcp translocation, and PKB activity.
TC Uptake-The initial uptake rate of TC in hepatocytes was determined as previously described (31). Briefly, at various times after incubation of hepatocytes with SB203580 and/or CPT-cAMP or hypotonic media, an aliquot of cell suspension (5-8 mg protein/ml) was withdrawn and used to determine the initial uptake rate of TC (20 M). Transport was initiated by adding cells to the incubation medium containing [ 14 C]TC and [ 3 H]inulin, with uptake determined at different time points. Initial uptake rates were calculated from the slope of the linear portion of time-dependent uptake curves and expressed in nmol/ min/mg of protein.
TC uptake in HuH-Ntcp cells was determined after incubation with 100 M CPT-cAMP (15 min) or in a hypotonic medium (25 min) by incubating with 20 M TC containing [ 3 H]TC (100 dpm/pmol) for 2 min. Cells were then washed twice with 0.5 ml of ice-cold HEPES buffer and lysed in 0.25 ml of a lysis buffer (20 mM Tris, 150 mM NaCl, 1% Triton, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, 10 g/ml aprotinin, 10 g/ml leupeptin, 500 nM okadaic acid, and 1 mM orthovanadate (pH 7.5). Aliquots of cell lysate were used to quantitate radioactivity, protein, and PKB activity. TC uptake in HuH-Ntcp was linear for at least 3 min, and the 2-min uptake was used to calculate initial TC uptake rate expressed in pmol/min/mg of protein.
Ntcp Translocation-To determine whether SB203580 affects cAMPand cell swelling-induced Ntcp translocation, hepatocytes were pretreated with 1 or 100 M SB203580 for 30 min before incubating with 10 M CPT-cAMP for 15 min or in a hypotonic medium for 25 min. Cell surface proteins were then biotinylated followed by separation of biotinylated proteins and detection of Ntcp using immunoblot analysis as described previously (23,24). A similar method was used to determine Ntcp translocation in HuH-Ntcp cells. Briefly, hepatocytes (or HuH-Ntcp cells) were washed twice in ice-cold phosphate-buffered saline (PBS; pH 8.0) and then exposed to sulfo-NHS-LC-Biotin (0.5 mg/ml, Pierce) in PBS for 1 h at 4°C followed by washing 3 times with excess PBS. Cell pellets were lysed by incubating in a lysis buffer for 1 h at 4°C, and the lysates were used to determine biotinylated and total Ntcp mass. To assay for biotinylated Ntcp, the lysates were incubated with streptavidin-agarose beads for 1 h. The beads were separated by centrifugation followed by washing with lysis buffer and then boiled in Laemmli sample buffer for 5 min followed by centrifugation. The resulting supernatant, containing biotinylated proteins, was subjected to immunoblot analysis to determine plasma membrane Ntcp.
For immunoblot analysis, proteins (10 -50 g) from whole cell lysates (total Ntcp) and supernatant containing biotinylated proteins (plasma membrane Ntcp) were subjected to 12% SDS-PAGE by the method of Laemmli (32) as previously described (33). Proteins were transferred electrophoretically from SDS gels to nitrocellulose membranes (Transblot, Transfer Membrane, 0.45 micron from Bio-Rad) and probed with the Ntcp antibody (1:2000 dilution). Peroxidase-conjugated anti-IgG was used as the secondary antibody. The immunoblots were developed with the Amersham ECL kit according to the manufacturer's instructions.
Protein Kinase Assay-Cell lysates obtained after various treatments of hepatocytes and HuH-Ntcp cells were assayed for PKB activity using commercially available assay kits. The assay of PKB involved immunoprecipitation of PKB followed by in vitro kinase assay using Crosstide as the substrate. PKB was immunoprecipitated by incubating 100 g of total cellular protein, and the immunocomplexes were incubated for 30 min at 30°C with 30 l of reaction mixture (kinase buffer containing 200 M ATP, 2 Ci of [␥-32 P]ATP and 100 M Crosstide). After the reaction, an aliquot (15 l) of the supernatant was transferred into Whatman p81 filter paper and washed 4 times with 3 ml of 175 mM phosphoric acid and once with distilled water. The filters were air-dried and then counted in a scintillation counter. PKB activity was expressed in pmol of PO 4 3Ϫ /min/mg of protein. For p38 MAP kinase, hepatocyte lysates (100 -150 g of total protein) were subjected to 10% SDS-PAGE. Separated proteins were transferred electrophoretically from SDS gels to nitrocellulose membranes and probed with the phospho-p38 MAP kinase (Thr 180 /Tyr 182 ) antibody (1: 1,000 dilution) to detect the activated form of p38 MAP kinase. The blot was stripped and reprobed with p38 MAP kinase antibody (1:1,000 dilution) to detect total p38 MAP kinase. The immunoblots were developed with an enhanced chemiluminescence kit (Amersham Biosciences) according to the manufacturer's instructions.
Other Methods-The Lowry method was used to determine cell protein (34). The blots were scanned in gray scale using Adobe Photoshop (Adobe System Inc., San Jose, CA), and the relative band densities were quantitated using Sigmal Gel (Jandel Scientific Software, San Rafael, CA). All values are expressed as the mean Ϯ S.E. Paired t test was used to statistically analyze data with p Ͻ 0.05 considered significant.

SB203580
Inhibits cAMP-induced Increases in PKB Activity, TC Uptake, and Ntcp Translocation-SB203580, a known inhibitor of p38 MAP kinase, has been shown to inhibit PKB in murine T cell line, CT6, and primary human T cells (25). To determine whether SB203580 also inhibits PKB activity in hepatocytes, we determined the concentration-dependent effect of SB203580 on p38 MAP kinase and PKB activity in cAMPstimulated hepatocytes. Cyclic AMP stimulated p38 MAP kinase activity by 50%, and this effect was inhibited completely by 1 M SB203580 (Fig. 1). In contrast, cAMP-stimulated PKB activity was not inhibited by 1 M SB203580. However, 10 and 100 M SB203580 inhibited significantly and completely, respectively, cAMP-induced increases in PKB activity (Fig. 1,  upper panel). SB202474, an inactive analogue of SB203580, did not affect either the basal or cAMP-stimulated PKB activity (data not shown). Thus, as in T cells, SB203580 inhibits PKB activity in hepatocytes at a concentration 10 times higher than the concentration needed to inhibit p38 MAP kinase. We also observed similar inhibition of hepatocyte growth factor-induced increases in p38 MAP kinase and PKB activity by SB203580 (data not shown). To distinguish the effects mediated via inhibition of p38 MAP kinase or PKB, further studies in hepatocytes were conducted using 1 and 100 M SB203580.
SB203580 did not inhibit basal TC uptake (Fig. 1, lower  panel). However, cAMP-induced increases in TC uptake were inhibited by 100 M but not by 1 M SB203580. Similar concentrations of SB202474 did not affect either basal or cAMPstimulated TC uptake (data not shown). Cyclic AMP-mediated increases in plasma membrane Ntcp were also inhibited by 100 M but not by 1 M SB203580 (Fig. 2). Thus, SB203580 appears to inhibit cAMP-mediated increases in TC uptake and Ntcp translocation by inhibiting PKB and not p38 MAP kinase.
Effect of SB203580 on Cell Swelling-induced Increases in PKB Activity, TC Uptake, and Ntcp Translocation-Cell swelling induced by exposing hepatocytes to hypotonic media has previously been shown to stimulate PI 3-kinase-dependent PKB activity and Ntcp translocation in hepatocytes (24). To determine whether cell swelling-induced increases in Ntcp translocation is mediated via PKB, we studied the effect of SB203580. Cell swelling increased PKB activity by 3-4-fold and P38 MAP kinase activity by 40 -60% (Fig. 3, upper panel). Although 1 M SB203580-inhibited cell swelling induced in-creases in p38 MAP kinase, but not PKB activity, 100 M SB203580 inhibited p38 MAP kinase as well as PKB activity. Cell swelling increased TC uptake by 2-fold as in a previous study (24), and this effect was inhibited by100 M, but not by 1 M SB203580 (Fig. 3, lower panel). Cell swelling also increased plasma membrane Ntcp, and this effect was inhibited by 100 M, but not by 1 M SB203580 (Fig. 4). Thus, the inhibitory effect of SB203580 appears to be mediated via PKB and not p38 MAP kinase.

DN-PKB Inhibits cAMP-and Cell Swelling-induced Increases in PKB Activity and TC Uptake in HuH-Ntcp Cells-To
further determine the role of PKB, studies were conducted in HuH-Ntcp cells transfected with DN-PKB. Sodium-dependent TC uptake has previously been shown in HuH-Ntcp cells (27). Studies in the presence and absence of extracellular Na ϩ showed that more than 90% of TC uptake was Na ϩ -dependent (350 Ϯ 25 versus 21 Ϯ 2.7 pmol/min/mg of protein; n ϭ 3). In addition, TC uptake was not affected in cells transfected with the empty vector.
Preliminary studies showed that cAMP and cell-swelling increased TC uptake and PKB activity in HuH-Ntcp cells. To determine the role of PKB, effects of cAMP and cell swelling on TC uptake and PKB activity were determined in HuH-Ntcp cells transfected with DN-PKB. Expression of DN-PKB was confirmed by immunoblot analysis. Cyclic AMP as well as cell swelling stimulated TC uptake by 50 -100% (Fig. 5, lower  panel) and PKB activity by 3-4-fold (Fig. 5, upper panel) in HuH-Ntcp cells. Basal TC uptake and PKB activity were not affected by DN-PKB (Fig. 5). However, cAMP-and cell swelling-induced increases in TC uptake and PKB activity were inhibited in cells transfected with DN-PKB. Cyclic AMP and cell swelling also increased plasma membrane Ntcp in HuH-Ntcp cells, and this effect was inhibited in cells transfected with DN-PKB (Fig. 6). These results indicate that cAMP-and cell swelling-induced Ntcp translocation to the plasma membrane is dependent on PKB activity.

Constitutively Active PKB (CA-PKB) Stimulates PKB Activity, TC Uptake, and Ntcp Translocation in HuH-Ntcp Cells-
The above studies would indicate that stimulation of TC uptake and Ntcp translocation by cAMP and cell swelling results from activation of PKB. In that case, activation of PKB independent of cAMP and cell swelling should also stimulate TC uptake and Ntcp translocation. This hypothesis was tested by determining TC uptake and Ntcp translocation in HuH-Ntcp cells transiently transfected with CA-PKB. Preliminary studies showed that transfection of HuH-Ntcp cells with 0.1 g of CA-PKB produced an inconsistent increase (0 -1-fold) in PKB activity. However, transfection with 0.5 and 1.0 g of CA-PKB resulted in 10-and 15-fold increases in PKB activity, respectively. Because cAMP and cell swelling increased PKB activity by 2-4fold in HuH-Ntcp cells (Fig. 5), further studies were conducted in cells transfected with 0.5 g of CA-PKB. Transfection with CA-PKB resulted in a 10-fold increase in PKB activity and a 65% increase in TC uptake (Fig. 7). Transfection with CA-PKB also increased plasma membrane Ntcp by 75% without affecting total Ntcp (Fig. 8). The increase in plasma membrane Ntcp is not due to an increase in Ntcp synthesis, since total Ntcp level was unaffected by CA-PKB. Thus, CA-PKB stimulates Ntcp translocation to the plasma membrane. These effects of CA-PKB transfection were not affected when PI3K was inhibited by wortmannin (Figs. 7 and 8), indicating that PKB is downstream of PI 3-kinase. These results support the hypothesis that PKB mediates cAMP and cell swelling-induced increases in TC uptake and Ntcp translocation. DISCUSSION The present study was designed to determine the role of PKB in cAMP-and cell swelling-induced increases in TC uptake and Ntcp translocation. Although previous studies (23,24) raised the possibility of a role for PKB, no direct evidence was presented. In the present study, the role of PKB was assessed using two different approaches, namely inhibitor and transfection studies. Results of these studies are consistent with a role for PKB as discussed below.
The first series of studies in hepatocytes showed that SB203580, a known inhibitor of p38 MAP kinase, inhibits cAMP-and cell swelling-induced increases in PKB activity at a concentration higher than that needed to inhibit p38 MAP kinase activity (Figs. 1 and 3). SB203580 has previously been shown to inhibit interleukin-2-stimulated PKB activity by inhibiting the PKB kinase, phosphoinositide-dependent protein kinase 1, in murine and human T cells (25). However, SB203580 does not inhibit insulin-stimulated PKB activity in 3T3-L1 adipocytes (35). Thus, inhibition of PKB activity by SB203580 may be cell-as well as stimulant-dependent.
Cyclic AMP and cell swelling activated p38 MAP kinase as well as PKB in hepatocytes. However, it is unlikely that cAMPand cell swelling-induced increases in TC uptake and Ntcp translocation is mediated via p38 MAP kinase, since 1 M SB203580, which inhibited p38 MAP kinase (Figs. 1 and 3), did not inhibit TC uptake and Ntcp translocation stimulated by cAMP and cell swelling (Figs. 1-4). On the other hand, inhibition of PKB activity by 100 M SB203580 was associated with inhibition of cAMP-and cell swelling-induced increases in TC uptake and Ntcp translocation. These results suggest that cAMP-and cell swelling-induced increases in TC uptake and Ntcp translocation may be mediated via PKB. It is, however, possible that the effect of SB203580 on TC uptake and Ntcp translocation is unrelated to its effect on PKB. Thus, studies were conducted in HuH-Ntcp cells transiently transfected with DN-PKB and CA-PKB to obtain more direct evidence in support of a role for PKB.
Our studies in HuH-Ntcp cells (Figs. 5 and 6) show that cAMP and cell swelling stimulate PKB, TC uptake, and Ntcp translocation. These results are similar to those observed in hepatocytes (23,24). The ability of cAMP and cell swelling to stimulate PKB was inhibited when HuH-Ntcp cells were transiently transfected with DN-PKB, and this was associated with inhibition of cAMP-and cell swelling-induced increases in TC uptake and Ntcp translocation ( Fig. 5 and 6). In addition, stimulation of PKB in HuH-Ntcp cells by CA-PKB resulted in stimulation of TC uptake and Ntcp translocation. These results indicate that the stimulation of PKB activity is required for cAMP-and cell swelling-induced increases in TC uptake and Ntcp translocation. The role of PKB in insulin-stimulated glucose transport and GLUT4 translocation has been supported by similar studies with constitutively active and dominant negative mutants of PKB in various cell types (5)(6)(7)(8)(9). Thus, stimulation of transporter translocation may be a general mechanism by which PKB regulates solute transport in various cell types.
Our previous studies show that cAMP-and cell swellinginduced increases in PKB activity are dependent on PI 3-kinase (23,24). Our present study showed that CA-PKB can stimulate TC uptake and Ntcp translocation when PI 3-kinase is inhibited by wortmannin (Figs. 7 and 8). Thus, the stimulation of Ntcp translocation by cAMP and cell swelling can be proposed to involve activation of PI 3-kinase followed by activation of PKB. The mechanism by which PKB stimulates Ntcp translocation is unclear. Because cAMP-mediated stimulation of Ntcp translocation is dependent on intact microfilament (23,36), it is possible that PKB stimulates microfilament-dependent movement of Ntcp-containing vesicles to the plasma membrane. PKB has been suggested to stimulate GLUT4 translocation by accelerating inter-endosomal transit of GLUT4-containing vesicle (9). Whether such a mechanism may also be involved in PKB-mediated Ntcp translocation cannot be evaluated at this time since intracellular vesicular compartments for Ntcp have not yet been definitely established.
A recent study shows that inhibition of p38 MAP kinase in 3T3-L1 adipocytes and L6 myotubules by SB203680 prevents insulin-stimulated glucose uptake but not GLUT4 translocation (35). The present study also showed that cAMP and cell swelling stimulate p38 MAP kinase in hepatocytes (Figs. 1 and  3). Because inhibition of p38 MAP kinase by 1 M SB203580 did not inhibit cAMP-or cell swelling-induced increases in TC uptake and Ntcp translocation, it is likely that neither TC uptake nor Ntcp translocation is mediated via a p38 MAPK kinase pathway. However, cAMP-stimulated p38 MAP kinase may be involved in cAMP-stimulated secretion of bile acids (37) and Bsep translocation (38). Recent studies suggest that tauroursodeoxycholate and cell swelling stimulate Bsep translocation via a p38 MAP kinase pathway (39). Thus, it is possible that cAMP-stimulated Bsep translocation is also mediated via a p38 MAP kinase pathway.
In conclusion, the present study strongly suggests that the stimulation of TC uptake and Ntcp translocation by cAMP and cell swelling is mediated via a PI 3-kinase/PKB signaling pathway in hepatocytes.