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(The FASEB Journal. 2006;20:2214-2222.)
© 2006 FASEB

Reduced intestinal and renal amino acid transport in PDK1 hypomorphic mice

Rexhep Rexhepaj*,1, Florian Grahammer*,1, Harald Völkl{dagger}, Christine Remy{ddagger}, Carsten A. Wagner{ddagger}, Diana Sandulache*, Ferruh Artunc*, Guido Henke*, Srinivas Nammi*, Giovambattista Capasso§, Dario R. Alessi|| and Florian Lang*,1,2

* Department of Physiology I, University of Tübingen, Germany;

{dagger} Department of Physiology, Medical University, Innsbruck, Austria;

{ddagger} Institute of Physiology and Center of Integrative Human Physiology, University of Zürich, Switzerland;

§ Chair of Nephrology, Second University of Napoli, Italy; and

|| Department of Biochemistry, University of Dundee, UK

2Correspondence: Department of Physiology, University of Tübingen Gmelinstr. 5, D-72076 Tübingen, Germany. E-mail: florian.lang{at}uni-tuebingen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The phosphoinositide-dependent kinase PDK1 activates the serum- and glucocorticoid-inducible kinase isoforms SGK1, SGK2, and SGK3 and protein kinase B, which in turn are known to up-regulate a variety of sodium-coupled transporters. The present study was performed to explore the role of PDK1 in amino acid transport. As mice completely lacking functional PDK1 are not viable, mice expressing 10–25% of PDK1 (pdk1hm) were compared with their wild-type (WT) littermates (pdk1wt). Body weight was significantly less in pdk1hm than in pdk1wtmice. Despite lower body weight of pdk1hmmice, food and water intake were similar in pdk1hm and pdk1wtmice. According to Ussing chamber experiments, electrogenic transport of phenylalanine, cysteine, glutamine, proline, leucine, and tryptophan was significantly smaller in jejunum of pdk1hmmice than in pdk1wt mice. Similarly, electrogenic transport of phenylalanine, glutamine, and proline was significantly decreased in isolated perfused proximal tubules of pdk1hmmice. The urinary excretion of proline, valine, guanidinoacetate, methionine, phenylalanine, citrulline, glutamine/glutamate, and tryptophan was significantly larger in pdk1hm than in pdk1wtmice. According to immunoblotting of brush border membrane proteins prepared from kidney, expression of the Na+-dependent neutral amino acid transporter B0AT1 (SLC6A19), the glutamate transporter EAAC1/EAAT3 (SLC1A1), and the transporter for cationic amino acids and cystine b0,+AT (SLC7A9) was decreased but the Na+/proline cotransporter SIT (SLC6A20) was increased in pdk1hm mice. In conclusion, reduction of functional PDK1 leads to impairment of intestinal absorption and renal reabsorption of amino acids. The combined intestinal and renal loss of amino acids may contribute to the growth defect of PDK1-deficient mice.—Rexhepaj, R., Grahammer, F., Völkl, H., Remy, C., Wagner, C. A., Sandulache, D., Artunc, F., Henke, G., Nammi, S., Capasso, G., Alessi, D. R., Lang, F. Reduced intestinal and renal amino acid transport in PDK1 hypomorphic mice.


Key Words: aminoaciduria • PI3 kinase • growth factors • SGK • PKB


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IN VITRO coexpression studies in Xenopus oocytes disclosed the ability of protein kinase B/Akt (1 , 2) and the serum- and glucocorticoid-inducible kinase family members SGK1 (3 4 5) , SGK2 (6) , and SGK3 (6) to up-regulate a variety of channels and transporters (7 8 9 10) , including the glutamine transporter SN1 (SLC38A3, SNAT3) (11) as well as glutamate transporters EAAT1 (SLCA3) (12) , EAAT2 (SCL1A2) (13) , EAAT3 (SLC1A1) (14) , EAAT4 (SLC1A6) (15) , and EAAT5 (SLC1A7) (16) .

PKB and SGKs are activated by IGF1 and insulin through the phosphatidylinositide 3 (PI3) kinase and phosphoinositide-dependent kinase PDK1 (17 18 19 20 21 22 23) . The PI3 kinase pathway is an integral element of growth factor, insulin, and IFN signaling (24 25 26 27 28 29) . Its pleotropic functions include regulation of cell survival (30 , 31) and cell proliferation (32 33 34 35) . Moreover, inactivation of PDK1 by the phosphatase PTEN is abrogated by oxidation, and thus PDK1 participates in the signaling of oxidative stress (36) . In view of its influence on the PKB and SGK isoforms, PDK1 may be a master switch in the growth factor-, insulin-, and stress-dependent regulation of amino acid transport.

The PDK1 knockout mouse is not viable (37) , highlighting the functional importance of this kinase. Mice with suppressed PDK1 activity of up to ~20% (pdk1hm) are significantly smaller than their age- and sex-matched WT littermates (pdk1wt) (37) . The smaller weight of the animals appeared to be primarily due to decreased cell volumes and not to cell number (37) . Among the determinants of cell volume is the concentrative cellular uptake of amino acids (38 39 40) . The present study was performed to elucidate the impact of PDK1 on transport of amino acids in the intestine and the kidney.

Several amino acid transport systems in mammals contribute to the intestinal absorption or renal proximal tubular reabsorption of amino acids (41 42 43) . Neutral amino acids are mainly transported by the Na+-dependent system B0 and IMINO encoded by the B0AT1 (SLC6A19) and SIT (SLC6A20) (43 44 45 46) . Accordingly, mutations in B0AT1 are responsible for Hartnup disease characterized by the impaired transport of neutral amino acids in the intestine and renal proximal tubule (47 , 48) . Anionic amino acids are transported in a Na+- and K+-dependent manner by the system X–AG EAAC1/EAAT3 (SLC1A1) transporter, whereas cationic amino acids and cystine are transported by the dimeric b0,+AT/rBAT (SLC7A9/SLC3A1) transporter. Mutations in either b0,+AT or rBAT cause cystinuria with reduced transport of these amino acids both in intestine and kidney (42 43 44 45 46 47 48 49) . So far, little if anything is known about the regulation of these transport systems in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Generation and basic properties of PDK1 hypomorphic mice have been described (37) . Genotyping was made by polymerase chain reaction (PCR) on tail DNA using PDK1 and neo-R-specific primers as described previously (37) . Mice had free access to standard mouse diet (C14310 Altromin, Langen, Germany) and tap water. All animal experiments were conducted according to the guidelines of The American Physiological Society, German law for the welfare of animals and were approved by the local authorities.

Food intake, fecal weight, and electrolyte composition
Mice were placed in individual metabolic cages (Tecniplast, Hohenpeissenberg, Germany). After a training period of 2 days, food and fluid intake as well as urinary and fecal output were determined under control conditions (control diet C1000 Altromin, Germany) over two consecutive 24 h periods. Results were averaged for each animal. The inner wall of the metabolic cages was siliconized and urine was collected under water-saturated oil to allow for quantitative measurements. Before and at the end of the metabolic cage experiments, ~150 µl blood was withdrawn into heparinized capillaries by puncturing the retro-orbital plexus. Hematocrit was determined after centrifugation. Plasma was separated for further analysis. Serum insulin-like growth factor (IGF) 1 was measured using an ELISA kit according to the manufacturer’s instructions (DSL-10–2900, dsl, Webster, TX, USA).

To determine amino acid concentrations in urine, mice were individually placed in metabolic cages and urine was collected over 24 h. To prevent bacterial growth and hence metabolism and breakdown of amino acids, 5 µl of concentrated acetic acid was added into the urine collectors beneath the mineral oil. Amino acid concentrations in urine and serum were measured by HPLC as described before (8) . Urinary creatinine concentrations were determined utilizing a commercial enzymatic kit (Labor und Technik, Berlin, Germany).

Electrogenic amino acid transport in isolated perfused proximal straight tubules
Experiments have been performed in proximal straight tubules. Segments of 0.2 to 0.4 mm length were dissected and perfused following principally the method of Burg et al. (50) . Modifications of the technique concerning track system, pipette arrangement, and use of dual channel perfusion pipettes have been described in detail previously (51 , 52) . The luminal perfusion rate was >10 nl/min. The bath was continuously perfused at a rate of 20 ml/min and thermostated with a dual channel feedback system (Hampel, Frankfurt, Germany). Bath temperature was kept constant at 38°C. The potential difference across the basolateral cell membrane (PDbl) was determined utilizing Ling-Gerard electrodes (100–200 M{Omega}) pulled from filament capillaries (1.5 o.d., 1.0 i.d., Hilgenberg, Malsfeld; Germany). The electrodes were connected to a high impedance electrometer (FD223, WPI, Science Trading, Frankfurt, Germany) via an Ag/AgCl half cell. An Ag/AgCl reference electrode was connected to the bath. Entry of positive charge by electrogenic transport is expected to depolarize the basolateral cell membrane. The magnitude of the depolarization depends on the magnitude of the induced current on the one hand and on the resistance of cell membranes and shunt on the other. PDbl was continuously recorded with and without L-phenylalanine, L-glutamine, or L-proline (20 mM each) in the luminal perfusate to stimulate electrogenic reabsorption as described (53) . The bath and luminal perfusates were composed of (all numbers mM): 110 NaCl, 5 KCl, 20 NaHCO3, 1.3 CaCl2, 1 MgCl2, and 2 Na2HPO4. In the bath, (in mM) 18 mannitol, 1 glucose (Glc), 1 glutamine, and 1 Na-lactate; 20 mannitol and 1 Ba2+ were added and in the lumen. Where indicated, 20 mM mannitol was replaced by 20 mM of the respective amino acid in the luminal perfusate. The bath solution was constantly gassed with a mixture of 95% O2 and 5% CO2, resulting in a pH of 7.4.

Ussing chamber experiments in small intestine
For analysis of electrogenic intestinal amino acid transport, animals were sacrificed, the abdomen was opened, and the intestine was quickly removed. Jejunal segments (5 to 10 cm postpylorus) were mounted into a custom-made mini-Ussing chamber with an opening diameter of 0.99 mm and an opening area of 0.00769 cm2. Under control conditions, the serosal and luminal perfusate contained (in mM): 115 NaCl, 2 KCl, 1 MgCl2, 1.25 CaCl2, 0.4 KH2PO4, 1.6 K2HPO4, 5 Na pyruvate, 25 NaHCO3, 20 mannitol (pH 7.4, adjusted with HCl). Where indicated, L-phenylalanine, L-cysteine, L-glutamine, L-proline, L-leucine, L-tryptophan, L-valine, L-methionine, and L-citrulline (20 mM, all from Roth, Karlsruhe, Germany) were added to the luminal perfusate at the expense of mannitol.

In all Ussing chamber experiments, the transepithelial potential difference (Vte) was determined continuously and transepithelial resistance (Rte) was estimated from the voltage deflections ({Delta}Vte) elicited by imposing test currents (It=1 µA). The resulting Rte and the short circuit current (Isc) were calculated according to Ohm’s law. All substances were from Sigma (Taufkirchen, Germany) or Roth (Karlsruhe, Germany).

Preparation of brush border membrane vesicles (BBMV)
BBMV were prepared from whole mouse kidney using the Mg2+ precipitation technique as described (54) After measurement of the total protein concentration (Bio-Rad Protein kit, Bio-Rad, Hercules, CA, USA), 20 µg of brush border membrane protein was solubilized in Laemmli sample buffer and SDS-PAGE was performed on 10% polyacrylamide gels. For immunoblotting, proteins were transferred electrophoretically from unstained gels to PVDF membranes (Immobilon-P, Millipore, Bedford, MA, USA). After blocking with 5% milk powder in Tris-buffered saline/0.1% Tween-20 for 60 min, the blots were incubated with affinity purified rabbit anti-B0AT1 (SLC6A19), rabbit anti-SIT (SLC6A20) (46) , rabbit anti-EAAC1/EAAT3 (SLC1A1) (Alpha Diagnostics, San Antonio, TX, USA), rabbit antib0,+AT1 (SLC7A9) antibodies (1:1000), and mouse monoclonal antiactin (42 kDa, Sigma) 1: 500 either for 2 h at room temperature or overnight at 4°C. After washing and subsequent blocking, blots were incubated with secondary antibodies conjugated with alkaline phosphatase or horseradish peroxidase (goat anti-rabbit 1:5000 and donkey anti-mouse 1:5000; Promega, Madison, WI, USA) for 1 h at room temperature. Antibody (Ab) binding was detected with the enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL, USA) in the case of HRP-linked antibodies and with the CDP Star kit (Roche, Nutley, NJ, USA) for activating protein (AP) linked antibodies before detection of chemiluminescence with the Diana III Chemiluminescence detection system. Bands were quantified with the Aida Image Analyzer software (Raytest, Straubenhardt, Germany).

Statistics
Data are provided as means ± SE; n represents the number of independent experiments. All data were tested for significance using the unpaired Student’s t test with Welch correction, where applicable, and only results with P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
As reported earlier, at the same age (32–36 wk) body weight was significantly smaller in pdk1hm mice (22.5±0.9 g, n=12) than in pdk1wt mice (32.7±1.3 g, n=12). Despite lower body weight of pdk1hm mice, food and water intake were similar in pdk1hm mice (3.8±0.2 g/24 h and 4.4±0.4 ml/24 h, respectively) as in pdk1wt mice (3.2±0.1 g/24 h and 4.2±0.5 ml/24 h, respectively). Consequently, if expressed per gram body weight, food and water intake were significantly larger in pdk1hm than pdk1wt mice (Fig. 1 ).


Figure 1
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  		Figure 1. Body weight, food and water intake (per 24h) in pdk1hm and pdk1wt mice. Arithmetic means ± SE (n=12) of body weight, food and water intake of PDK1 hypomorphic mice (pdk1hm, filled columns) and WT littermates (pdk1wt, open columns). *Statistically significant difference between pdk1hm and pdk1wt mice.

In theory, growth retardation could result from defective release of growth hormone leading to decreased formation of IGF1. To explore this possibility, we determined plasma IGF-1 concentrations in both weaning (18 days) and adult (4 months) animals. However, the respective values were not significantly different between pdk1hm mice (338±43 ng/ml and 454±44 ng/ml, respectively, n=7–9) and pdk1wt mice (332±13 ng/ml and 483±64 ng/ml, respectively, n=7–9).

Additional experiments were performed to determine whether altered weight was paralleled by altered intestinal transport. To determine PDK1-dependent amino acid transport, segments of jejunum from pdk1hm and pdk1wt mice were mounted into mini-Ussing chambers and electrogenic amino acid transport was determined utilizing electrophysiological analysis (Fig. 2 ). In the absence of luminal substrates, the transepithelial potential difference (Vte) of jejunal segments amounted to –4.17 ± 0.33 mV (n=13) in pdk1hm mice and to –4.11 ± 0.24 mV (n=14) in pdk1wt mice. The transepithelial resistance (Rte) approached 9.4 ± 0.7 {Omega} · cm2 (n=13) in pdk1hm mice and 8.3 ± 0.4 {Omega} · cm2 (n=14) in pdk1wt mice. Neither transepithelial potential difference nor transepithelial resistance were significantly different between pdk1hm and pdk1wt mice. Accordingly, calculated basal short circuit current (Isc, basal) was not different between the two genotypes, reaching –472 ± 56 µA/cm2 in pdk1hm mice and –514 ± 46 µA/cm2 in pdk1wt mice, respectively.


Figure 2
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  		Figure 2. Amino acid-induced short circuit current (Isc,aa) in proximal jenunal segments. Alterations of transepithelial voltages ({Delta}Vaa) and induced short circuit currents (Isc,aa) in proximal segments of jejunal tissue from PDK1 hypomorphic mice (pdk1hm) and WT littermates (pdk1wt) before and after addition of phenylalanine (Phe), cysteine (Cys), glutamine (Gln), proline (Pro), leucine (Leu), tryptophan (Try), methionine (Met), valine (Val), and citrulline (Cit). A) Original tracings illustrating the effect of amino acids on the transepithelial potential difference. B) Arithmetic means ± SE (n=13–14) of amino acid-induced short circuit currents in jejunum from pdk1hm mice (open columns) and pdk1wt mice (filled columns). * Statistically significant difference between pdk1hm and pdk1wt mice.

The iso-osmotic replacement of mannitol by phenylalanine, cysteine, glutamine, proline, or leucine created a lumen-negative shift of the transepithelial potential difference ({Delta}Vaa) without significantly altering the transepithelial resistance. The {Delta}Vaa and Rte allowed calculation of the amino acid-induced short circuit current (Isc,aa). The Isc,aa was smaller in pdk1hm than in pdk1wt mice, a difference reaching statistical significance for phenylalanine, cysteine, glutamine, proline, leucine, and tryptophan (Table 1 , Fig. 2 ). The currents induced by methionine, valine, and citrulline tended to be lower in pdk1hm mice, a difference that did not reach statistical significance between the genotypes, however (Table 1 , Fig. 2 ).


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  		Table 1. Amino acid induced currents in jejunuma

Similar to intestine, proximal renal tubules display decreased electrogenic transport of amino acids in PDK1hm mice. As illustrated in Fig. 3 , the potential difference across the basolateral cell membrane (PDbl) of isolated perfused straight proximal tubules (i.e., late parts of proximal tubule) was in the absence of amino acids not significantly different between pdk1hm mice (–51.4±2.5, n=13) and pdk1wt mice (–54.4±1.5 mV, n=14). Addition of 20 mM L-phenylalanine, L-glutamine, or L-proline, respectively, to the luminal fluid significantly decreased PDbl in both pdk1hm and pdk1wt mice, an effect significantly smaller in pdk1hm than in pdk1wt mice (Fig. 3) .


Figure 3
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  		Figure 3. Effect of amino acids on the potential difference across the basolateral cell membrane of straight proximal tubules. A, B) Original tracings illustrating the effect of the luminal application of 20 mM phenylalanine (Phe), glutamine (GLN), and proline (PRO) in the presence of the K+ channel blocker Ba2+ (1 mM) on the potential difference across the basolateral cell membrane of straight proximal tubules (PDbl) from PDK1 hypomorphic mice (pdk1hm) and WT littermates (pdk1wt). C) Arithmetic means ± SE of the depolarization of the basolateral cell membrane from pdk1hm mice (open columns) and pdk1wt mice (filled columns) following the luminal replacement of 20 mM mannitol with 20 mM phenylalanine (n=5 pdk1hm and 6 pdk1wt), glutamine (n=5 pdk1hm and 5 pdk1wt) or proline (n=3 pdk1hm and 3 pdk1wt). *Statistically significant difference between pdk1hm and pdk1wt mice.

To assess the abundance of major amino acid transporter proteins expressed in the brush border membrane of the proximal tubule, immunoblotting was performed with isolated brush border membranes from kidney. A reduced abundance of the major renal Na+-dependent amino acid transporter for neutral amino acids, B0AT1 (SLC6A19), was found (Fig. 4 ). In parallel, the abundance of the Na+-dependent glutamate transporter EAAC1/EAAT3 (SLC1A1) was decreased. Expression of the b0,+AT (SLC7A9) protein, the catalytic subunit of system b0,+ responsible for the reabsorption of cationic amino acids and cystine, was also reduced. However, expression of the Na+, proline cotransporter SIT (SLC6A20) was enhanced in the pdk1hm kidney. Thus, at least in the kidney, decreased expression of several major renal amino acid transporters contributes to the impaired amino acid reabsorption in PDK1 hypomorphic mice.


Figure 4
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  		Figure 4. Abundance of amino acid transporters in kidney brush border membrane. Upper panel) Immunoblots for b0,+AT (SLC7A9), SIT (SLC6A20), EAAC1 (SLC1A1), B0AT1 (SLC6A19) amino acid transporters, and actin demonstrate the significant reduction in protein abundance of b0,+AT, EAAC1, B0AT1, and increased expression of SIT amino acid transporters in the renal brush border membrane of pdk1hm mice. Lower panel) Bar graphs summarizing quantification of amino acid transporter abundance normalized for loading with actin (ratio transporter/actin). *Statistically significant differences between pdk1wt (open columns) and pdk1hm (open columns) mice (n=5 for each genotype).

More experiments were performed to elucidate the plasma concentrations and renal excretion of the amino acids. As evident from Table 2 , plasma concentration of none of the amino acids was significantly different between pdk1hm and pdk1wt mice. Table 3 shows the urinary excretion of creatinine and amino acids. The average daily urinary creatinine excretion was not significantly different between pdk1hm mice (17.6±1.2 µg/24 h/g body wt) and pdk1wt mice (15.7±0.7 µg/24 h/g body wt). To account for individual variations of urinary concentration, the daily urinary excretion of individual amino acids was divided by the respective daily creatinine excretion. As indicated in Table 3 , renal excretions of several amino acids were larger in pdk1hm than in pdk1wt mice, differences reaching statistical significance for proline, valine, guanidinoacetate, methionine, phenylalanine, citrulline, glutamine/glutamate, and tryptophan.


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  		Table 2. Plasma concentrations of amino acids in pdk1hm and pdk1wt micea


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  		Table 3. Urinary excretion of amino acids in pdk1hm and pdk1wt micea


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
As reported (37) , PDK1 hypomorphic mice (pdk1hm) are significantly smaller than their age- and sex-matched WT littermates (pdk1wt). Evidence suggested that the decrease of body mass is the result of smaller cell volumes and is not due to a decrease in cell number (37) . In theory, a decrease of cell volume could be due to lack of nutrients, as concentrative uptake of amino acids leads to cell swelling and subsequent stimulation of protein synthesis (38 39 40 , 55) . Accordingly, the decreased cell volume could be due to impaired nutrient uptake.

Moreover, insufficient dietary supply or defective renal or intestinal uptake of amino acids is typically paralleled by delayed growth (56 57 58 59 60) . Thus, the impaired intestinal uptake and renal retention of amino acids could contribute to the growth defect of the PDK1-deficient mice. On the other hand, food and water intake was not decreased in PDK1-deficient mice, further highlighting the significance of impaired intestinal absorption.

According to the Ussing chamber experiments, electrogenic transport of phenylalanine, cysteine, glutamine, proline, leucine, and tryptophan is impaired. Previous in vitro studies revealed the stimulating effect of the SGK isoforms and/or PKB on the amino acid transporters SN1 (SLC38A3, SNAT3) (11) , ASCT2 (SLC1A5) (61) , EAAT1 (SLC1A3) (12) , EAAT2 (SLC1A2) (13) , EAAT3 (SLC1A1) (14) , EAAT4 (SLC1A6) (15) , and EAAT5 (SLC1A7) (16) . It is possible that other transporters may also be targets of these kinases. The profile of amino acids in urine and the reduction observed in transport-induced currents for several amino acids point to the involvement of more than one amino acid transport system (41) . Patients with mutations in either B0AT1 (SLC6A19) or b0,+AT (SLC7A9) and rBAT (SLC3A1) suffer from Hartnup disorder or cystinuria. Both diseases are characterized by the impaired renal and intestinal transport of neutral amino acids such as phenylalanine and leucine or cationic amino acids and cystine, respectively (47 , 48 , 62 , 63) Accordingly, the reduction in B0AT1 (SLC6A19) expression correlates with the loss of leucine, phenylalanine, and glutamine. Na-dependent leucine absorption occurs in the kidney via several systems, including the low-affinity system B0 (B0AT1, SLC6A19) in the initial part of the proximal tubule and probably related but unidentified members of the same gene family. XT2 (SLC6A18) and XT3, two related orphan transporters with no clearly established transport function (46) , have been observed in the late proximal tubule. The electrophysiological measurements in the late proximal tubule indicated that amino acid-induced currents were smaller in the pdk1hm mice, suggesting that not only a reduction of B0AT1 (SLC6A19) expression in the initial proximal tubule but also reduced function (and/or expression) of these putative amino acid transporters may contribute to the observed aminoaciduria and reduced currents. The relatively high urinary loss of methionine and valine may result from the fact that no compensatory mechanisms exist. In contrast, the increased abundance of the SIT transporter may point to a compensatory mechanism. SIT (SLC6A20) is mainly expressed in the late proximal tubule; it appears to transport particularly imino amino acids and belongs to the same SLC6 family of amino acid transporters as B0AT1 (SLC6A19) (44 45 46) . Surprisingly, reduced abundance of the b0,+AT (SLC7A9) subunit of system b0,+ did not induce urinary loss of its typical substrates arginine, lysine, and cystine. Again, the observed decrease in protein abundance may not adequately mirror the actual activity in the brush border membrane. Other transport systems that may compensate for loss of b0,+ activity are presently unknown. The increased abundance of SIT may indicate that regulation of amino acid transporters by PDK1 does not affect all amino acid transporters.

The SGK kinase isoforms stimulate the voltage-gated K+ channel complex KCNE1/KCNQ1 (64) , which contributes to maintenance of the potential difference across the apical cell membrane of the renal proximal tubule (53 , 65) , a critical driving force for electrogenic amino acid transport (66 , 67) . K+ channels similarly tune intestinal transport of amino acids (68) . The SGK kinase isoforms further stimulate the Na+/K+-ATPase (69 70 71 72) , which is required to maintain the chemical driving force for Na+ coupled nutrient transport (68) . Thus, decreased PDK1 activity could modify nutrient transport indirectly by compromising the driving forces. The potential difference across the basolateral cell membrane of proximal renal tubules was, however, not significantly different between pdk1hm and pdk1wt mice. Moreover, a decreased K+ channel activity should enhance and not decrease the depolarization following addition of substrates for Na+ coupled transport. Thus, the blunted depolarization in pdk1hm mice reflects decreased electrogenic amino acid transport rather than decreased K+ channel activity.

PDK1 may not only stimulate the transport of amino acids, but may participate in the regulation of further nutrients. SGK1 has been shown to stimulate activity of the Na+-glucose cotransporter SGLT1 (SLC5A1) (73) and the facilitative Glc transporter GLUT1 (SLC2A1) (74) . It has also been shown to stimulate the Na+, glucose dicarboxylate cotransporter NaDC-1 (SLC13A2) (75) and the creatine transporter CreaT (SLC6A8) (76) .

Any impairment of renal electrolyte excretion by blunted stimulation through PDK1 may be compensated for by enhanced stimulation through other mechanisms. In fact, despite the powerful stimulating effect of SGK1 on the renal epithelial Na+ channel ENaC (77 , 78) , complete knockout of SGK1 leads to only moderate impairment of renal Na+ retention, which is disclosed only after exposure to a salt-deficient diet (79) .

The mild reduction in transport rates observed here may be explained by the only moderate decrease in transport function compared with the loss of function mutations or knockout leading to severe loss of the respective amino acids (47 , 48 , 62 , 63 , 80) . It should be kept in mind that the mice still express PDK1, and thus that PDK1-dependent regulation of amino acid transport is not completely disrupted in those mice.

The defective intestinal and renal transport of amino acids did not lead to gross alterations of plasma amino acid concentrations. Apparently, the enhanced food intake per body weight compensates for the renal loss of amino acids and maintains extracellular amino acid concentrations sufficient for cellular uptake. Needless to say, the availability of amino acids in extracellular fluid does not preclude impairment of cellular amino acid uptake in PDK1-deficient animals through modification of amino acid transport in nonpolarized cells.

In conclusion, the PDK1 hypomorphic mice display moderate impairment of amino acid transport, which presumably contributes to the delayed growth of those mice. The observations described here disclose a novel player in the regulation of intestinal and renal nutrient transport.


   ACKNOWLEDGMENTS
 
This work was supported by the European Commission (LSHM-CT-2003–502852; EUGINDAT) to C.A.W., G.C., and F.L., and the Deutsche Forschungsgemeinschaft (La 315/4–6, GRK 1302) to F.L. and D.A. S.N. was a scholar of the German Academic Exchange Service (DAAD). The authors acknowledge the meticulous preparation of the manuscript by Lejla Subasic and Tanja Loch.


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication January 5, 2006. Accepted for publication June 12, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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