'Reduced Intestinal and Renal Amino Acid Transport in PDK1

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 (2006). Reduced intestinal and renal amino acid transport in PDK1 hypomorphic mice. FASEB Journal , 20(13):2214-2222. Postprint available at: http://www.zora.uzh.ch

University of Zurich Posted at the Zurich Open Repository and Archive, University of Zurich. Zurich Open Repository and Archive http://www.zora.uzh.ch

Originally published at: FASEB Journal 2006, 20(13):2214-2222. Winterthurerstr. 190 CH-8057 Zurich http://www.zora.uzh.ch

Year: 2006

Reduced intestinal and renal amino acid transport in PDK1 hypomorphic 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

Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch

Originally published at: FASEB Journal 2006, 20(13):2214-2222. Reduced intestinal and renal amino acid transport in PDK1 hypomorphic mice

Abstract

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 (pdk1(hm)) were compared with their wild-type (WT) littermates (pdk1(wt)). Body weight was significantly less in pdk1(hm) than in pdk1(wt) mice. Despite lower body weight of pdk1(hm) mice, food and water intake were similar in pdk1(hm) and pdk1(wt) mice. According to Ussing chamber experiments, electrogenic transport of phenylalanine, cysteine, glutamine, proline, leucine, and tryptophan was significantly smaller in jejunum of pdk1(hm) mice than in pdk1(wt) mice. Similarly, electrogenic transport of phenylalanine, glutamine, and proline was significantly decreased in isolated perfused proximal tubules of pdk1(hm) mice. The urinary excretion of proline, valine, guanidinoacetate, methionine, phenylalanine, citrulline, glutamine/glutamate, and tryptophan was significantly larger in pdk1(hm) than in pdk1(wt) mice. According to immunoblotting of brush border membrane proteins prepared from kidney, expression of the Na+-dependent neutral amino acid transporter B(0)AT1 (SLC6A19), the glutamate transporter EAAC1/EAAT3 (SLC1A1), and the transporter for cationic amino acids and cystine b(0,+)AT (SLC7A9) was decreased but the Na+/proline cotransporter SIT (SLC6A20) was increased in pdk1(hm) 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. The FASEB Journal • Research Communication

Reduced intestinal and renal amino acid transport in PDK1 hypomorphic mice

Rexhep Rexhepaj,*,1 Florian Grahammer,*,1 Harald Vo¨lkl,† Christine Remy,‡ Carsten A. Wagner,‡ Diana Sandulache,* Ferruh Artunc,* Guido Henke,* ʈ Srinivas Nammi,* Giovambattista Capasso,§ Dario R. Alessi, and Florian Lang*,1,2 *Department of Physiology I, University of Tu¨bingen, Germany; †Department of Physiology, Medical University, Innsbruck, Austria; ‡Institute of Physiology and Center of Integrative Human Physiology, University of Zu¨rich, Switzerland; §Chair of Nephrology, Second University of Napoli, Italy; and ʈ Department of Biochemistry, University of Dundee, UK

ABSTRACT The phosphoinositide-dependent kinase IN VITRO coexpression studies in Xenopus oocytes PDK1 activates the serum- and glucocorticoid-inducible disclosed the ability of protein kinase B/Akt (1, 2) and kinase isoforms SGK1, SGK2, and SGK3 and protein the serum- and glucocorticoid-inducible kinase family kinase B, which in turn are known to up-regulate a members SGK1 (3–5), SGK2 (6), and SGK3 (6) to variety of sodium-coupled transporters. The present up-regulate a variety of channels and transporters (7– study was performed to explore the role of PDK1 in 10), including the glutamine transporter SN1 amino acid transport. As mice completely lacking func- (SLC38A3, SNAT3) (11) as well as glutamate transport- tional PDK1 are not viable, mice expressing 10–25% of ers EAAT1 (SLCA3) (12), EAAT2 (SCL1A2) (13), PDK1 (pdk1hm) were compared with their wild-type EAAT3 (SLC1A1) (14), EAAT4 (SLC1A6) (15), and (WT) littermates (pdk1wt). Body weight was significantly EAAT5 (SLC1A7) (16). less in pdk1hm than in pdk1wtmice. Despite lower body PKB and SGKs are activated by IGF1 and insulin weight of pdk1hmmice, food and water intake were through the phosphatidylinositide 3 (PI3) kinase and similar in pdk1hm and pdk1wtmice. According to Ussing phosphoinositide-dependent kinase PDK1 (17–23). chamber experiments, electrogenic transport of phe- The PI3 kinase pathway is an integral element of nylalanine, cysteine, glutamine, proline, leucine, and growth factor, insulin, and IFN signaling (24–29). Its tryptophan was significantly smaller in jejunum of pleotropic functions include regulation of cell survival pdk1hmmice than in pdk1wtmice. Similarly, electrogenic (30, 31) and cell proliferation (32–35). Moreover, transport of phenylalanine, glutamine, and proline was inactivation of PDK1 by the phosphatase PTEN is significantly decreased in isolated perfused proximal abrogated by oxidation, and thus PDK1 participates in hm tubules of pdk1 mice. The urinary excretion of pro- the signaling of oxidative stress (36). In view of its line, valine, guanidinoacetate, methionine, phenylala- influence on the PKB and SGK isoforms, PDK1 may be nine, citrulline, glutamine/glutamate, and tryptophan a master switch in the growth factor-, insulin-, and hm wt was significantly larger in pdk1 than in pdk1 mice. stress-dependent regulation of amino acid transport. According to immunoblotting of brush border mem- The PDK1 knockout mouse is not viable (37), high- brane proteins prepared from kidney, expression of ؉ lighting the functional importance of this kinase. Mice the Na -dependent neutral amino acid transporter with suppressed PDK1 activity of up to ϳ20% (pdk1hm) 0 B AT1 (SLC6A19), the glutamate transporter EAAC1/ are significantly smaller than their age- and sex- EAAT3 (SLC1A1), and the transporter for cationic wt ؉ matched WT littermates (pdk1 ) (37). The smaller amino acids and cystine b0, AT (SLC7A9) was de- ؉ weight of the animals appeared to be primarily due to creased but the Na /proline cotransporter SIT decreased cell volumes and not to cell number (37). hm (SLC6A20) was increased in pdk1 mice. In conclusion, Among the determinants of cell volume is the concen- reduction of functional PDK1 leads to impairment of trative cellular uptake of amino acids (38–40). The intestinal absorption and renal reabsorption of amino present study was performed to elucidate the impact of acids. The combined intestinal and renal loss of amino PDK1 on transport of amino acids in the intestine and acids may contribute to the growth defect of PDK1- the kidney. deficient mice.—Rexhepaj, R., Grahammer, F., Vo¨lkl, H., Several amino acid transport systems in mammals Remy, C., Wagner, C. A., Sandulache, D., Artunc, F., contribute to the intestinal absorption or renal proxi- Henke, G., Nammi, S., Capasso, G., Alessi, D. R., Lang, F. Reduced intestinal and renal amino acid transport in PDK1 hypomorphic mice. FASEB J. 20, 2214–2222 (2006) 1 These authors contributed equally to this work. 2 Correspondence: Department of Physiology, University of Tu¨bingen Gmelinstr. 5, D-72076 Tu¨bingen, Germany. E-mail: Key Words: aminoaciduria ⅐ PI3 kinase ⅐ growth factors fl[email protected] ⅐ SGK ⅐ PKB doi: 10.1096/fj.05-5676com

2214 0892-6638/06/0020-2214 © FASEB mal tubular reabsorption of amino acids (41–43). Modiﬁcations of the technique concerning track system, Neutral amino acids are mainly transported by the pipette arrangement, and use of dual channel perfusion ϩ Na -dependent system B0 and IMINO encoded by the pipettes have been described in detail previously (51, 52). The luminal perfusion rate was Ͼ10 nl/min. The bath was B0AT1 (SLC6A19) and SIT (SLC6A20) (43–46). Ac- 0 continuously perfused at a rate of 20 ml/min and thermo- cordingly, mutations in B AT1 are responsible for stated with a dual channel feedback system (Hampel, Frank- Hartnup disease characterized by the impaired trans- furt, Germany). Bath temperature was kept constant at 38°C. port of neutral amino acids in the intestine and renal The potential difference across the basolateral cell mem- proximal tubule (47, 48). Anionic amino acids are brane (PDbl) was determined utilizing Ling-Gerard elec- ϩ ϩ ⍀ transported in a Na - and K -dependent manner by trodes (100–200 M ) pulled from ﬁlament capillaries (1.5 Ϫ the system X EAAC1/EAAT3 (SLC1A1) trans- o.d., 1.0 i.d., Hilgenberg, Malsfeld; Germany). The electrodes AG were connected to a high impedance electrometer (FD223, porter, whereas cationic amino acids and cystine are 0,ϩ WPI, Science Trading, Frankfurt, Germany) via an Ag/AgCl transported by the dimeric b AT/rBAT (SLC7A9/ half cell. An Ag/AgCl reference electrode was connected to 0,ϩ SLC3A1) transporter. Mutations in either b AT or the bath. Entry of positive charge by electrogenic transport is rBAT cause cystinuria with reduced transport of these expected to depolarize the basolateral cell membrane. The amino acids both in intestine and kidney (42–49). So magnitude of the depolarization depends on the magnitude far, little if anything is known about the regulation of of the induced current on the one hand and on the resistance of cell membranes and shunt on the other. PD was contin- these transport systems in vivo. bl uously 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 MATERIALS AND METHODS bath and luminal perfusates were composed of (all numbers

mM): 110 NaCl, 5 KCl, 20 NaHCO3, 1.3 CaCl2, 1 MgCl2, and Animals 2Na2HPO4. In the bath, (in mM) 18 mannitol, 1 glucose (Glc), 1 glutamine, and 1 Na-lactate; 20 mannitol and 1 Ba2ϩ Generation and basic properties of PDK1 hypomorphic mice were added and in the lumen. Where indicated, 20 mM have been described (37). Genotyping was made by polymer- mannitol was replaced by 20 mM of the respective amino acid ase chain reaction (PCR) on tail DNA using PDK1 and in the luminal perfusate. The bath solution was constantly neo-R-specific primers as described previously (37). Mice had gassed with a mixture of 95% O2 and 5% CO2, resulting in a pH free access to standard mouse diet (C14310 Altromin, Lan- of 7.4. gen, Germany) and tap water. All animal experiments were conducted according to the guidelines of The American Ussing chamber experiments in small intestine Physiological Society, German law for the welfare of animals and were approved by the local authorities. For analysis of electrogenic intestinal amino acid transport, Food intake, fecal weight, and electrolyte composition 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 Mice were placed in individual metabolic cages (Tecniplast, chamber with an opening diameter of 0.99 mm and an Hohenpeissenberg, Germany). After a training period of 2 opening area of 0.00769 cm2. Under control conditions, the days, food and fluid intake as well as urinary and fecal output serosal and luminal perfusate contained (in mM): 115 NaCl, were determined under control conditions (control diet 2 KCl, 1 MgCl , 1.25 CaCl , 0.4 KH PO , 1.6 K HPO ,5Na C1000 Altromin, Germany) over two consecutive 24 h peri- 2 2 2 4 2 4 pyruvate, 25 NaHCO , 20 mannitol (pH 7.4, adjusted with ods. Results were averaged for each animal. The inner wall of 3 HCl). Where indicated, l-phenylalanine, l-cysteine, l-glu- the metabolic cages was siliconized and urine was collected tamine, l-proline, l-leucine, l-tryptophan, l-valine, l-methio- under water-saturated oil to allow for quantitative measure- nine, and l-citrulline (20 mM, all from Roth, Karlsruhe, ments. Before and at the end of the metabolic cage experi- Germany) were added to the luminal perfusate at the expense ments, ϳ150 ␮l blood was withdrawn into heparinized capil- of mannitol. laries by puncturing the retro-orbital plexus. Hematocrit was In all Ussing chamber experiments, the transepithelial determined after centrifugation. Plasma was separated for potential difference (V ) was determined continuously and further analysis. Serum insulin-like growth factor (IGF) 1 was te transepithelial resistance (R ) was estimated from the voltage measured using an ELISA kit according to the manufacturer’s te deflections (⌬V ) elicited by imposing test currents (I ϭ1 instructions (DSL-10–2900, dsl, Webster, TX, USA). te t ␮A). The resulting R and the short circuit current (I ) were To determine amino acid concentrations in urine, mice te sc calculated according to Ohm’s law. All substances were from were individually placed in metabolic cages and urine was Sigma (Taufkirchen, Germany) or Roth (Karlsruhe, Ger- collected over 24 h. To prevent bacterial growth and hence many). metabolism and breakdown of amino acids, 5 ␮l of concen- trated acetic acid was added into the urine collectors beneath the mineral oil. Amino acid concentrations in urine and serum Preparation of brush border membrane vesicles (BBMV) were measured by HPLC as described before (8). Urinary creatinine concentrations were determined utilizing a commer- BBMV were prepared from whole mouse kidney using the cial enzymatic kit (Labor und Technik, Berlin, Germany). Mg2ϩ precipitation technique as described (54) After mea- surement of the total protein concentration (Bio-Rad Protein Electrogenic amino acid transport in isolated perfused kit, Bio-Rad, Hercules, CA, USA), 20 ␮g of brush border proximal straight tubules membrane protein was solubilized in Laemmli sample buffer and SDS-PAGE was performed on 10% polyacrylamide gels. Experiments have been performed in proximal straight tu- For immunoblotting, proteins were transferred electro- bules. Segments of 0.2 to 0.4 mm length were dissected and phoretically from unstained gels to PVDF membranes (Im- perfused following principally the method of Burg et al. (50). mobilon-P, Millipore, Bedford, MA, USA). After blocking

REDUCED INTESTINAL AND RENAL AMINO ACID TRANSPORT 2215 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 Diagnos- tics, San Antonio, TX, USA), rabbit antib0,ϩAT1 (SLC7A9) antibodies (1:1000), and mouse monoclonal antiactin (42 kDa, Sigma) 1: 500 either for2hatroom 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) for1hatroom 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, Strauben- hardt, Germany).

Statistics

Data are provided as means Ϯ se; n represents the number of independent experiments. All data were tested for signiﬁ- cance using the unpaired Student’s t test with Welch correction, where applicable, and only results with P Ͻ 0.05 were considered statistically signiﬁcant.

RESULTS

As reported earlier, at the same age (32–36 wk) body weight was significantly smaller in pdk1hm mice (22.5Ϯ0.9 g, nϭ12) than in pdk1wt mice (32.7Ϯ1.3 g, nϭ12). Despite lower body weight of pdk1hm mice, food and water intake were similar in pdk1hm mice (3.8Ϯ0.2 g/24 h and 4.4Ϯ0.4 ml/24 h, respectively) as in pdk1wt mice (3.2Ϯ0.1 g/24 h and 4.2Ϯ0.5 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). 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 wean- ing (18 days) and adult (4 months) animals. However, the respective values were not significantly different between pdk1hm mice (338Ϯ43 ng/ml and 454Ϯ44 ng/ml, respectively, nϭ7–9) and pdk1wt mice (332Ϯ13 ng/ml and 483Ϯ64 ng/ml, respectively, nϭ7–9). 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 cham- bers and electrogenic amino acid transport was determined utilizing electrophysiological analysis (Fig. 2). In the absence of luminal substrates, the transepithelial Figure 1. Body weight, food and water intake (per 24h) in pdk1hmand pdk1wt mice. Arithmetic means Ϯ se (nϭ12) of potential difference (Vte) of jejunal segments amounted to –4.17 Ϯ 0.33 mV (nϭ13) in pdk1hm mice body weight, food and water intake of PDK1 hypomorphic mice (pdk1hm, filled columns) and WT littermates (pdk1wt, and to –4.11 Ϯ 0.24 mV (nϭ14) in pdk1wt mice. The Ϯ ⍀ open columns). *Statistically significant difference between transepithelial resistance (Rte) approached 9.4 0.7 pdk1hm and pdk1wt mice. ⅐ cm2 (nϭ13) in pdk1hm mice and 8.3 Ϯ 0.4 ⍀ ⅐ cm2

2216 Vol. 20 November 2006 The FASEB Journal REXHEPAJ ET AL. Figure 2. Amino acid-induced short circuit current (Isc,aa) in proximal jenunal segments. Alterations of transepithelial voltages ⌬ hm ( Vaa) and induced short circuit currents (Isc,aa) in proximal segments of jejunal tissue from PDK1 hypomorphic mice (pdk1 ) 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 Ϯ se (nϭ13–14) 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.

ϭ wt (n 14) in pdk1 mice. Neither transepithelial poten- difference across the basolateral cell membrane (PDbl) tial difference nor transepithelial resistance were signif- of isolated perfused straight proximal tubules (i.e., late icantly different between pdk1hm and pdk1wt mice. parts of proximal tubule) was in the absence of amino Accordingly, calculated basal short circuit current acids not signiﬁcantly different between pdk1hm mice Ϯ ϭ wt Ϯ (Isc, basal) was not different between the two genotypes, (–51.4 2.5, n 13) and pdk1 mice (–54.4 1.5 mV, reaching –472 Ϯ 56 ␮A/cm2 in pdk1hm mice and nϭ14). Addition of 20 mM l-phenylalanine, l-glu- –514 Ϯ 46 ␮A/cm2 in pdk1wt mice, respectively.

The iso-osmotic replacement of mannitol by phenyl- a alanine, cysteine, glutamine, proline, or leucine cre- TABLE 1. Amino acid induced currents in jejunum ated a lumen-negative shift of the transepithelial poten- ⌬ Current in Current in tial difference ( Vaa) without significantly altering the wt hm ⌬ Amino acid pdk1 pdk1 n transepithelial resistance. The Vaa and Rte allowed calculation of the amino acid-induced short circuit Phenylalanine –373 Ϯ 33 –245 Ϯ 44* 13–14 hm Ϯ Ϯ current (Isc,aa). The Isc,aa was smaller in pdk1 than in Cysteine –389 47 –243 35* 13–14 pdk1wt mice, a difference reaching statistical signifi- Glutamine –447 Ϯ 62 –284 Ϯ 49* 13–14 Proline –367 Ϯ 30 –254 Ϯ 36* 13–14 cance for phenylalanine, cysteine, glutamine, proline, Ϯ Ϯ leucine, and tryptophan (Table 1, Fig. 2). The currents Leucine –697 57 –423 35* 5 Tryptophan –260 Ϯ 20 –196 Ϯ 19* 6 induced by methionine, valine, and citrulline tended to Ϯ Ϯ hm Methionine –435 44 –334 35 6 be lower in pdk1 mice, a difference that did not reach Valine –568 Ϯ 90 –462 Ϯ 70 6 statistical significance between the genotypes, however Citrulline –478 Ϯ 110 –333 Ϯ 56 6 (Table 1, Fig. 2). a Ϯ ␮ 2 Similar to intestine, proximal renal tubules display Arithmetic means se of the currents (in A/cm ) generated by the respective amino acids (20 mm). *Statistically significant decreased electrogenic transport of amino acids in difference between pdk1hm and pdk1wt mice, n ϭ number of mice PDK1hm mice. As illustrated in Fig. 3, the potential studied.

REDUCED INTESTINAL AND RENAL AMINO ACID TRANSPORT 2217 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 Kϩ channel blocker Ba2ϩ (1 mM) on the potential difference across the basolateral cell membrane of straight proximal tubules hm wt Ϯ (PDbl) from PDK1 hypomorphic mice (pdk1 ) and WT littermates (pdk1 ). 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 (nϭ5 pdk1hm and 6 pdk1wt), glutamine (nϭ5 pdk1hm and 5 pdk1wt) or proline (nϭ3 pdk1hm and 3 pdk1wt). *Statistically significant difference between pdk1hm and pdk1wt mice. tamine, or l-proline, respectively, to the luminal fluid ing statistical significance for proline, valine, guanidi- hm wt significantly decreased PDbl in both pdk1 and pdk1 noacetate, methionine, phenylalanine, citrulline, glu- mice, an effect significantly smaller in pdk1hm than in tamine/glutamate, and tryptophan. pdk1wt mice (Fig. 3). To assess the abundance of major amino acid transporter proteins expressed in the brush border mem- DISCUSSION brane of the proximal tubule, immunoblotting was performed with isolated brush border membranes from As reported (37), PDK1 hypomorphic mice (pdk1hm) ϩ kidney. A reduced abundance of the major renal Na - are significantly smaller than their age- and sex- dependent amino acid transporter for neutral amino matched WT littermates (pdk1wt). Evidence suggested acids, B0AT1 (SLC6A19), was found (Fig. 4). In paral- that the decrease of body mass is the result of smaller ϩ lel, the abundance of the Na -dependent glutamate cell volumes and is not due to a decrease in cell number transporter EAAC1/EAAT3 (SLC1A1) was decreased. (37). In theory, a decrease of cell volume could be due ϩ Expression of the b0, AT (SLC7A9) protein, the cata- to lack of nutrients, as concentrative uptake of amino ϩ lytic subunit of system b0, responsible for the reab- acids leads to cell swelling and subsequent stimulation sorption of cationic amino acids and cystine, was also of protein synthesis (38–40, 55). Accordingly, the ϩ reduced. However, expression of the Na , proline decreased cell volume could be due to impaired nutri- cotransporter SIT (SLC6A20) was enhanced in the ent uptake. pdk1hm kidney. Thus, at least in the kidney, decreased Moreover, insufficient dietary supply or defective expression of several major renal amino acid transport- renal or intestinal uptake of amino acids is typically ers contributes to the impaired amino acid reabsorp- paralleled by delayed growth (56–60). Thus, the im- tion in PDK1 hypomorphic mice. paired intestinal uptake and renal retention of amino More experiments were performed to elucidate the acids could contribute to the growth defect of the plasma concentrations and renal excretion of the PDK1-deficient mice. On the other hand, food and amino acids. As evident from Table 2, plasma concen- water intake was not decreased in PDK1-deficient mice, tration of none of the amino acids was significantly further highlighting the significance of impaired intes- different between pdk1hm and pdk1wt mice. Table 3 tinal absorption. shows the urinary excretion of creatinine and amino According to the Ussing chamber experiments, elec- acids. The average daily urinary creatinine excretion trogenic transport of phenylalanine, cysteine, glu- was not significantly different between pdk1hm mice tamine, proline, leucine, and tryptophan is impaired. (17.6Ϯ1.2 ␮g/24 h/g body wt) and pdk1wt mice Previous in vitro studies revealed the stimulating effect of (15.7Ϯ0.7 ␮g/24 h/g body wt). To account for individ- the SGK isoforms and/or PKB on the amino acid trans- ual variations of urinary concentration, the daily uri- porters SN1 (SLC38A3, SNAT3) (11), ASCT2 (SLC1A5) nary excretion of individual amino acids was divided by (61), EAAT1 (SLC1A3) (12), EAAT2 (SLC1A2) (13), the respective daily creatinine excretion. As indicated EAAT3 (SLC1A1) (14), EAAT4 (SLC1A6) (15), and in Table 3, renal excretions of several amino acids were EAAT5 (SLC1A7) (16). It is possible that other trans- larger in pdk1hm than in pdk1wt mice, differences reach- porters may also be targets of these kinases. The profile

2218 Vol. 20 November 2006 The FASEB Journal REXHEPAJ ET AL. 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 measure- ments 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) (44–46). Surprisingly, reduced abundance of the ϩ ϩ 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 ϩ that may compensate for loss of b0, activity are pres- ently 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 ϩ K channel complex KCNE1/KCNQ1 (64), which con-

Figure 4. Abundance of amino acid transporters in kidney TABLE 2. Plasma concentrations of amino acids in pdk1hmand brush border membrane. Upper panel) Immunoblots for pdk1wt micea b0,ϩAT (SLC7A9), SIT (SLC6A20), EAAC1 (SLC1A1), B0AT1 (SLC6A19) amino acid transporters, and actin demonstrate pdk1 wt pdk1 hm the significant reduction in protein abundance of b0,ϩAT, EAAC1, B0AT1, and increased expression of SIT amino acid Ϯ Ϯ hm Glycine 275.4 29.4 279.8 32.7 transporters in the renal brush border membrane of pdk1 Alanine 398.9 Ϯ 64.7 375.7 Ϯ 54.8 mice. Lower panel) Bar graphs summarizing quantification of Aminobutyrate 23.2 Ϯ 2.7 18.9 Ϯ 1.6 amino acid transporter abundance normalized for loading Serine 38.7 Ϯ 5.9 34.9 Ϯ 5.4 with actin (ratio transporter/actin). *Statistically significant Ϯ Ϯ wt hm Proline 53.2 11.7 66.2 16.1 differences between pdk1 (open columns) and pdk1 Valine 223.2 Ϯ 28.8 269.5 Ϯ 36.5 ϭ (open columns) mice (n 5 for each genotype). Threonine 31.3 Ϯ 6.0 37.3 Ϯ 6.7 5-Oxoproline 15.9 Ϯ 1.6 16.5 Ϯ 1.3 Leucineϩisoleucine 216.0 Ϯ 23.7 250.0 Ϯ 32.8 of amino acids in urine and the reduction observed in Asparagine 34.2 Ϯ 4.3 43.3 Ϯ 5.6 transport-induced currents for several amino acids Methionine 71.5 Ϯ 11.1 83.2 Ϯ 11.4 point to the involvement of more than one amino acid Histidine 33.9 Ϯ 2.2 32.5 Ϯ 1.7 transport system (41). Patients with mutations in either Citrulline 53.4 Ϯ 4.3 55.6 Ϯ 4.5 ϩ B0AT1 (SLC6A19) or b0, AT (SLC7A9) and rBAT Phenylalanine 110.8 Ϯ 12.8 118.2 Ϯ 16.7 Methylhistidine 44.3 Ϯ 2.6 43.6 Ϯ 2.3 (SLC3A1) suffer from Hartnup disorder or cystinuria. Ϯ Ϯ Both diseases are characterized by the impaired renal Tyrosine 140.9 21.2 138.2 17.1 Aspartate 23.7 Ϯ 3.7 24.7 Ϯ 4.5 and intestinal transport of neutral amino acids such as Glutamate/glutamine 71.5 Ϯ 8.1 60.3 Ϯ 8.3 phenylalanine and leucine or cationic amino acids and Tryptophan 20.1 Ϯ 2.2 18.2 Ϯ 2.2 cystine, respectively (47, 48, 62, 63) Accordingly, the Aminoadipate 5.1 Ϯ 0.9 6.8 Ϯ 1.3 reduction in B0AT1 (SLC6A19) expression correlates N-Acetylasparaginic acid 2.1 Ϯ 0.4 1.6 Ϯ 0.3 with the loss of leucine, phenylalanine, and glutamine. Ornithine 49.2 Ϯ 6.4 70.4 Ϯ 12.8 Na-dependent leucine absorption occurs in the kidney a Ϯ ϭ 0 Arithmetic means se (n 11) of the individual amino acid via several systems, including the low-affinity system B concentrations in plasma (␮m) from PDK1 hypomorphic (pdk1hm) (B0AT1, SLC6A19) in the initial part of the proximal mice and wild type littermates (pdk1wt).

REDUCED INTESTINAL AND RENAL AMINO ACID TRANSPORT 2219 TABLE 3. Urinary excretion of amino acids in pdk1hmand 78), complete knockout of SGK1 leads to only moder- wt a ϩ pdk1 mice ate impairment of renal Na retention, which is disclosed only after exposure to a salt-deficient diet (79). wt hm pdk1 pdk1 The mild reduction in transport rates observed here may be explained by the only moderate decrease in Glycine 496.3 Ϯ 86.4 526.5 Ϯ 110.7 Alanine 143.8 Ϯ 22.9 208.9 Ϯ 110.7 transport function compared with the loss of function Proline 17.0 Ϯ 2.7 34.3 Ϯ 10.6* mutations or knockout leading to severe loss of the Valine 19.4 Ϯ 2.3 27.5 Ϯ 3.7* respective amino acids (47, 48, 62, 63, 80). It should be Guanidinoacetate 301.7 Ϯ 32.3 414.6 Ϯ 52.0* kept in mind that the mice still express PDK1, and thus Leucine 153.5 Ϯ 78.3 140.7 Ϯ 73.2 that PDK1-dependent regulation of amino acid trans- Ϯ Ϯ Creatine 2286.5 263.4 2476.4 272.6 port is not completely disrupted in those mice. Ornithine 5.6 Ϯ 1.4 6.4 Ϯ 1.4 Lysine 21.3 Ϯ 6.7 15.6 Ϯ 5.0 The defective intestinal and renal transport of amino Methionine 53.4 Ϯ 8.4 94.1 Ϯ 20.0* acids did not lead to gross alterations of plasma amino Histidine 8.2 Ϯ 1.4 15.3 Ϯ 6.7 acid concentrations. Apparently, the enhanced food Phenylalanine 11.1 Ϯ 1.3 17.6 Ϯ 2.5* intake per body weight compensates for the renal loss Arginine 8.2 Ϯ 2.3 11.8 Ϯ 2.9 of amino acids and maintains extracellular amino acid Citrulline 8.5 Ϯ 1.9 14.2 Ϯ 3.0* Ϯ Ϯ concentrations sufficient for cellular uptake. Needless Tyrosine 28.9 2.6 45.6 10.7 to say, the availability of amino acids in extracellular Glutamine/glutamate 53.9 Ϯ 7.6 105.3 Ϯ 25.2* Tryptophan 4.4 Ϯ 0.5 6.8 Ϯ 1.5* fluid does not preclude impairment of cellular amino 5-Hydroxytryphtophane 0.43 Ϯ 0.2 0.7 Ϯ 0.2 acid uptake in PDK1-deficient animals through modifi- Cystine 7.17 Ϯ 1.5 9.1 Ϯ 1.9 cation of amino acid transport in nonpolarized cells. Homocystine 0.20 Ϯ 0.14 0.26 Ϯ 0.13 In conclusion, the PDK1 hypomorphic mice display moderate impairment of amino acid transport, which aArithmetic means Ϯ se (nϭ12–14) of the urinary excretion of individual amino acid excretion (in mmol/mol creatinine) from presumably contributes to the delayed growth of those PDK1-deficient (pdk1hm) mice and wild type littermates (pdk1wt). mice. The observations described here disclose a novel *Statistically significant difference between pdk1hm and pdk1wt mice. player in the regulation of intestinal and renal nutrient transport. tributes to maintenance of the potential difference This work was supported by the European Commission across the apical cell membrane of the renal proximal (LSHM-CT-2003–502852; EUGINDAT) to C.A.W., G.C., and tubule (53, 65), a critical driving force for electrogenic F.L., and the Deutsche Forschungsgemeinschaft (La 315/ ϩ amino acid transport (66, 67). K channels similarly 4–6, GRK 1302) to F.L. and D.A. 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