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HORMONES AND SIGNALING

Intestinal function of gene-targeted mice lacking serum- and glucocorticoid-inducible kinase 1

Departments of ¹Physiology, ²Internal Medicine, and ³Anatomy, University of Tübingen, Tübingen; ⁴Department of Clinical Neurobiology, University of Heidelberg, Heidelberg; and ⁵Department of Biology, Chemistry, and Pharmacy, Free University Berlin, Berlin, Germany

Submitted 23 May 2005 ; accepted in final form 4 January 2006

	ABSTRACT

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In vitro experiments have revealed the ability of serum- and glucocorticoid-inducible kinase 1 (SGK1) to stimulate intestinal Na⁺-coupled glucose cotransporter 1 (SGLT1) and intestinal Na⁺/H⁺ exchanger 3 (NHE3). The present study explored the contribution of SGK1 to the regulation of intestinal transport in vivo. SGK1 transcript levels were determined by real-time PCR and glucose-induced currents (I_g) reflecting SGLT1 activity by Ussing chamber experiments. BCECF fluorescence was utilized for the determination of Na⁺-dependent pH recovery from an ammonium pulse (pH_NHE) reflecting NHE activity. As a result, intestinal SGK1 transcript levels were significantly enhanced by a 4-day treatment with 10 µg·mg body wt^–1·day^–1 dexamethasone (Dex). I_g was, under control conditions, virtually identical in sgk1 knockout mice (sgk1^–/–) and their wild type littermates (sgk1^+/+). A 4-day treatment with Dex, however, increased I_g approximately threefold in sgk1^+/+ mice but not in sgk1^–/– mice. pH_NHE was similar in sgk1^–/– and sgk1^+/+ mice before treatment. Dex increased pH_NHE approximately threefold in sgk1^+/+ mice and approximately twofold in sgk1^–/–mice, an effect significantly blunted in the presence of the specific NHE3 blocker S-3226 (10 µM). According to Western blot analysis, Dex significantly enhanced SGLT1 and NHE3 protein abundance in brush-border membranes of sgk1^+/+ mice but not of sgk1^–/–mice. In conclusion, basic functions of SGLT1 and NHE3 in the intestine do not require stimulation by SGK1. However, the effects of glucocorticoids on SGLT1 are fully, and on NHE3 partially, dependent on SGK1.

glucocorticoids; glucose transport; Na⁺-coupled glucose transporter 1; Na⁺/H⁺ exchanger3; intestine

SGK1 expression is particularly abundant in the intestine (17, 59, 60), suggesting a role of the kinase in intestinal transport regulation. Coexpression of SGK1 in Xenopus oocytes indeed upregulates a number of epithelial channels and transporters (41), such as the epithelial Na⁺ channel (ENaC) (7, 12, 15, 22, 42, 46, 50, 56, 58, 61), the renal outer medullary K⁺ channel (ROMK1) (47, 48, 67), and the voltage-gated K⁺ channel complex KCNE1/KCNQ1 (21). In addition to channels, SGK1 regulates Na⁺/H⁺ exchanger 3 (NHE3) (65, 66), the glutamine transporter SN1 (11), the glutamate transporter EAAT1 (10), renal and intestinal Na⁺-coupled glucose transporter 1 (SGLT1) (19), and Na⁺-K⁺-ATPase (27, 53, 56, 68).

Hitherto, nothing is known about how these in vitro observations translate into physiological regulation of intestinal function. The present study was thus performed to elucidate whether the regulation of intestinal transport is dependent on the expression of SGK1. To this end, SGLT1 and NHE3 activity were compared in gene-targeted mice lacking SGK1 (sgk1^–/– mice) and their wild-type littermates (sgk1^+/+ mice). SGLT1 activity was estimated from the glucose-induced equivalent short-circuit current (I_sc) and NHE3 activity from the Na⁺-dependent and S-3226-sensitive cytosolic pH recovery from an ammonium pulse. In part of the experiments, animals were treated with the glucocorticoid dexamethasone to stimulate SGK1 transcription.

	METHODS

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Animals. As described previously (64), a conditional targeting vector was generated from a 7-kb fragment encompassing the entire transcribed region on 12 exons. The neomycin resistance cassette was flanked by two LoxP sites and inserted into intron 11. Exons 4–11, which code for the Sgk1 kinase domain, were "floxed" by inserting a third LoxP site into intron 3. Targeted R1 ES cells were transiently transfected with Cre recombinase. A clone with a recombination between the first and third LoxP site (type I recombination) was injected into C57BL/6 blastocytes. Male chimeras were bred to 129/SvJ females. Heterozygous sgk1-deficient mice were backcrossed to 129/SvJ wild-type mice for two generations and then intercrossed to generate homozygous sgk1^–/– mice and sgk1^+/+ littermates. The animals were genotyped by PCR using standard methods. All animal experiments were conducted following the guidelines of the American Physiological Society and were approved by local authorities according to German law for the care and use of laboratory animals.

Dexamethasone treatment. For the analysis of dexamethasone effects, sgk1^+/+ and sgk1^–/– mice were injected with dexamethasone phosphate disodium salt (Sigma; Taufkirchen, Germany; dissolved in 0.9% saline) at a dose of 10 µg/g body wt for 4 consecutive days at 8 PM. Sgk1^–/– and sgk1^+/+ mice injected with 0.9% saline alone served as controls. Mice had free access to a standard mouse diet (Altromin diet 1310; Heidenau, Germany) and tap water. On the 4th day of dexamethasone treatment, animals were fasted for 4 h before the experiments with free access to tap water.

Quantitative real-time PCR measurements. To obtain intestinal tissues, animals were deeply anesthetized with diethylether and killed by cervical dislocation, and the abdomen was opened. The intestine was quickly removed and carefully flushed with 4°C control buffer to remove remaining food particles. Specific intestinal segments were rapidly frozen in liquid nitrogen. Automated disruption and homogenization of frozen tissue was performed using the MagNa Lyser Instrument (Roche Diagnostics; Mannheim, Germany). For each sample, one-way special tubes were filled with ceramic beads, 20–30 mg of frozen tissue, and 600 µl of RLT buffer (Qiagen; Hilden, Germany). Cleared cell lysate was transferred for further RNA purification (RNAeasy Mini Kit, Qiagen). Subsequently, 1 µg of total RNA was reverse transcribed to cDNA utilizing the reverse transcription system (Bioscience) with oligo(dT) primers according to the manufacturer's protocol. To determine mRNA levels, quantitative real-time PCR with the LightCycler System (Roche Diagnostics) was established. PCRs were performed in a final volume of 20 µl containing 2 µl cDNA, 2.4 µl MgCl₂ (3 µM), 1 µl primer mix (0.5 µM of both primers), 2 µl cDNA Master SYBR green I mix (Roche Molecular Biochemicals), and 12.6 µl DEPC-treated water. The transcript levels of the housekeeping gene mouse (m)GAPDH were also determined for each sample using a commercial primer kit (Search LC; Heidelberg, Germany). PCRs for GAPDH were performed in a final volume of 20 µl containing 2 µl cDNA, 2 µl primer mix (Search LC), 2 µl cDNA Master SYBR green I mix (Roche Molecular Biochemicals), and 14 µl DEPC-treated water.

Amplification of target DNA was performed during 35 cycles of 95°C for 10 s, 68°C for 10 s, and 72°C for 16 s, each with a temperature transition rate of 20°C/s and a secondary target temperature of 58°C with a step size of 0.5°C.

For mSGK3, the same protocol was used with the difference that the annealing temperature was 62–53°C and the number of amplification cycles was increased to 45.

Melting curve analysis was performed at 95°C for 0 s, 58°C for 10 s, and 95°C for 0 s to determine the melting temperatures of primer dimers and the specific PCR products. Melting curve analysis confirmed the amplified products, which were then separated on 1.5% agarose gels to confirm the expected size (406 bp mSGK1, 413 bp mSGK2, and 208 bp for mSGK3). Finally, results were calculated as a ratio of target versus housekeeping gene transcripts.

The following primers were used: mSGK1(GenBank No. NM_011361), sense 5'-TGT CTT GGG GCT GTC CTG TAT G-3' and antisense 5'-GCT TCT GCT GCT TCC TTC ACA C-3'; mSGK2 (GenBank No. NM_013731), sense 5'-CCA CAG ACT TTG ATT TCC TC-3' and antisense 3'-GGC AGT CCA AGA GAA TGT T-5'; and mSGK3 (GenBank No. NM_133220), sense 5'-ATGCAGAGGGATTGTATCATGG-3' and antisense 5'-GAGCCATAGCAGGAAACTGC-3'.

Brush-border membrane preparation. Small intestines were rinsed with ice-cold saline solution and opened longitudinally. Briefly, the mucosa was scraped off with a glass slide in a buffer containing (in mM) 250 sucrose, 20 Tris (pH 7.5), and 5 EGTA with a protease inhibitor cocktail (Roche). The suspension was homogenized in a Dounce homogenizer for 2 min. MgCl₂ was added to the homogenate to a final concentration of 10 mM. The suspension was stirred on ice and then centrifuged at 1,600 g for 15 min. Plasma membranes retained in the supernatant were collected by centrifugation at 20,000 g for 30 min. The resultant pellet was suspended in a pH 7.4 buffer consisting of (in mM) 125 sucrose, 10 Tris (pH 7.5), 2.5 EGTA, and 2.5 MgSO₄. This suspension was homogenized with 50 up-down strokes with a glass homogenizer and centrifuged at 20,000 g for 30 min. The final pellet, containing the purified brush-border membrane vesicles (BBMVs), was homogenized by passing the suspension through 25- and 28-gauge needles and solubilized. All the steps were carried out at 4°C. After the final suspension, samples were frozen at –80°C for later use. Membrane protein was assessed as described by Bradford.

Antibodies. Blots were incubated with an antibody against mSGLT1 (AAF17249 [GenBank] ) using a rabbit polyclonal antibody (kindly donated by Dr. Koepsell, Würzburg, Germany) raised against the synthetic peptide corresponding to amino acids 586–601, KDTIEIDTEAPQKKKG. The antibody against NHE3 was purchased from Alpha Diagnostics.

SDS-PAGE and Western blot analysis. Similar amounts of protein (50 µg) of BBMVs were solubilized in Laemmli sample buffer and resolved by 8% SDS-PAGE. Proteins were electrotransferred onto nitrocellulose membranes for 1 h at constant voltage of 100 V and blocked for 3 h in 5% nonfat milk in PBS-0.15% Tween 20 at room temperature.

The membrane was incubated with 1:2,000 anti-rabbit SGLT1 and 1:500 anti-rat NHE3 overnight at 4°C followed by goat anti-rabbit IgG coupled to horseradish peroxidase (1:1,000, Amersham Biosciences; Freiburg, Germany). After each incubation, the membrane was washed extensively with PBS-0.15% Tween 20 and then visualization with enhanced chemiluminescence, which was performed according to the manufacture's instructions (Amersham). Densitometric analysis of SGLT1 and NHE3 was performed using Bio-Rad Quantity One software (Bio-Rad; Munich, Germany)

In situ hybridization of SGK-1 mRNA. Adult mice were killed by carbon dioxide incubation. The large and small intestines were removed, immediately frozen in –25°C cold isopentane, and sliced on a freezing microtome at 12 µm thickness. Sections were subsequently mounted on silane-coated slides [2% 3-aminopropyltriethoxy-silane (Sigma) in acetone], dried at 60°C for 30 s, and fixed with 4% phosphate-buffered paraformaldehyde for 20 min. After three washes with PBS (0.1 M, pH 7.4), slides were incubated with Tris-EDTA buffer (100 mM Tris and 50 mM EDTA; pH 8) containing 2 µg/ml proteinase K for 10 min at room temperature and rinsed again three times with PBS. To reduce nonspecific background, slides were acetylated with triethanolamine buffer (0.1 M triethanolamine; pH 8.0) containing 0.25% (vol/vol) acetic anhydride (Sigma) twice for 5 min. After prehybridization with hybridization buffer [50% formamide (Sigma), 10% dextran sulfate, 5 mM EDTA, 20 mM Tris (pH 8), 10 mM DTT, 1x Denhardt's solution, 0.05% tRNA, and 300 mM NaCl] for 1 h at 62°C, sections were incubated with fresh hybridization buffer containing the denatured DIG-labeled sense or antisense probe (200 ng/ml) overnight at 63°C. After hybridization, slides were briefly rinsed in 2x SSC at room temperature and three times in 0.1x SSC for 15 min at 63°C. Detection of the DIG-labeled RNA probe was performed according to the protocol of the DIG nucleic acid detection kit (Roche; Basel, Switzerland). Tissues were blocked for 30 min with blocking buffer [1% blocking reagent (Roche)] in maleic acid buffer (0.1 M maleic acid and 0.15 M NaCl; pH 7.5) and then incubated with alkaline phosphatase-conjugated antibody solution [anti-DIG antibody (1:2,500, Roche)] in blocking buffer containing 0.1% Triton X-100 for 1 h. After being washed four times with maleic acid buffer for 15 min, slides were equilibrated for 5 min in Tris buffer [0.1 M Tris (pH 9.5), 0.1 M NaCl, and 50 mM MgCl₂]. The color development was carried out with freshly prepared substrate solution [nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate (X-phosphate; Roche) in Tris buffer (pH 9.5)]. After being washed three times with PBS, slides were rinsed in distilled water, dried, and placed on a coverslip with Kaiser's solution (Merck; Darmstadt, Germany).

Intestinal SGLT1 activity. For the analysis of electrogenic intestinal glucose transport, proximal (5–10 cm postpylorus) and distal (15–20 cm postpylorus) jejunal segments were mounted into a custom-made mini-Ussing chamber with an opening of 0.07 cm². Under control conditions, the serosal and luminal perfusate for jejunal experiments contained (in mM) 95 NaCl, 2 KCl, 1 MgCl₂, 1.25 CaCl₂, 0.4 KH₂PO₄, 1.6 K₂HPO₄, 5 Na pyruvic acid, 25 NaHCO₃, and 40 mannitol (pH 7.4, NaOH) with 1 µM indomethacin (Sigma) to prevent PGE₂-stimulated Cl^– secretion due to preparation of the intestine. Where indicated, glucose (10, 20, or 40 mM) was added to the luminal perfusate at the expense of mannitol.

In all Ussing chamber experiments, the transepithelial potential difference (V_t) was determined continuously, and transepithelial resistance (R_t) was estimated from the voltage deflections (V_t) elicited by imposing test currents. The resulting R_t and I_sc were calculated according to Ohm's law.

All substances were from Sigma or Roth (Karlsruhe, Germany).

Intestinal NHE3 activity. For the isolation of ileal villi, animals were fasted for 6 h before the experiments. After the animals were euthanized, the terminal 2 cm of the ileum were removed and cut longitudinally. After being washed with standard HEPES solution, the intestine was sliced into 0.3-cm² sections. Tissues were transferred onto the cooled stage of a dissecting microscope, and individual villi were detached from the intestine by snapping of the ileal base using sharpened microdissection tweezers. Care was taken not to touch the apical part of the villi. The villi were attached to a glass coverslip precoated with Cell-Tak adhesive (BD Biosciences).

For quantitative digital imaging of intracellular pH (pH_i), isolated individual villi were incubated in a HEPES-buffered Ringer solution containing 10 µM BCECF-AM (Molecular Probes; Leiden, The Netherlands) for 15 min at 37°C. After the chamber had been loaded, it was flushed for 5 min with Ringer solution to remove any deesterified dye sticking to the outside of the villi. The perfusion chamber was mounted on the stage of an inverted microscope (Zeiss Axiovert 135), which was used in the epifluorescence mode with a x40 oil-immersion objective (Zeiss Neoplan). BCECF was successively excited at 490/10 and 440/10 nm, and the resultant fluorescent signal was monitored at 535/10 nm using an intensified charge-coupled device camera (Proxitronic) and specialized computer software (Metafluor). Individual cells from the brush border of the villi were outlined and monitored during the course of the measurement. Intensity ratio data (490/440 nm) were converted into pH values using the high-K⁺/nigericin calibration technique (25).

The solutions, flow lines, and perfusion chamber were maintained at 37°C by a thermostatically controlled heating system. The volume of the perfusion chamber was 600 µl, and the flow rate was 4 ml/min for all solutions. For acid loading, cells were transiently exposed to a solution containing 20 mM NH₄Cl, leading to marked initial alkalinization of pH_i due to entry of NH₃ and binding of H⁺ to form NH₄⁺ (52).The acidification of pH_i upon the removal of ammonia allowed us to calculate the mean intrinsic buffering power of the cells (52), assuming that NH₄⁺ and NH₃ are in equilibrium in cytosolic and extracellular fluid and that ammonia leaves the cells as NH₃

where pH_i is the decrease of pH_i after ammonia removal and [NH₄⁺]_i is the decrease of the cytosolic NH₄⁺ concentration, which is identical to the concentration of NH₄⁺ immediately before the removal of ammonia. Given the pK for NH₄⁺/NH₃ of 8.9 (13), an extracellular pH (pH_o) of 7.4, and the NH₄⁺ concentration in extracellular fluid ([NH₄⁺]_o) of 19.37, [20/(1+10^{pH_o – pK})]: [NH₄⁺]_i = 19.37 x 10^{pH_o – pH_i}.

For some experiments, the selective NHE3 inhibitor S-3226 (Aventis; Frankfurt, Germany) was added to all solutions at a concentration of 10 µM.

Standard HEPES solution was composed of (in mM) 115 NaCl, 5 KCl, 1 CaCl₂, 1.2 MgSO₄, 2 NaH₂PO₄, 10 glucose, and 32.2 HEPES. Sodium-free HEPES solution was composed of (in mM) 132.8 NMDG, 3 KCl, 1 CaCl₂, 1.2 MgSO₄, 2 KH₂PO₄, 32 HEPES, 10 mannitol, and 10 glucose. For the sodium-free ammonium chloride solution, 10 NMDG and 10 mannitol were replaced with 20 mM NH₄Cl. The high-K⁺ solution for calibration was composed of (in mM) 105 KCl, 1 CaCl₂, 1.2 MgSO₄, 32.2 HEPES, and 10 mannitol (5 µM nigericin). The pH of the solutions was titrated to 7.4 or 7.0 with HCl/NaOH, HCl/NMDG, and HCl/KOH, respectively, at 37°C.

Statistics. Data are provided as means ± SE; n represents the number of independent experiments. All data were tested for significance using Student's t-test or ANOVA (Dunnet's test) where applicable, and only results with P < 0.05 were considered statistically significant.

	RESULTS

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Intestinal SGK1 expression under dexamethasone treatment. We used quantitative RT-PCR to test whether mSGK1 transcription is upregulated by a 4-day treatment with dexamethasone in different intestinal segments of wild-type mice. After the dexamethasone treatment of those mice, the mSGK1-to-mGAPDH ratio x1,000 increased in the jejunum from 12.3 ± 3.6 (n = 6) to 31.5 ± 6.6 (n = 7) and in the ileum from 32.6 ± 12.9 (n = 6) to 119.9 ± 22.7 (n = 7) (Fig. 1). Hence, a 4-day treatment with the glucocorticoid dexamethasone consistently increased intestinal mSGK1 transcript levels. As was expected, no mSGK1 transcripts were detected in SGK1 knockout mice (SGK1-to-GAPDH ratio 0.0, n = 4).

View larger version (8K): [in this window] [in a new window]   Fig. 1. Serum- and glucocorticoid-inducible kinase 1 (SGK1) transcript levels in intestinal segments. Ratios of the transcript levels of mouse (m)sgk1 over GAPDH in the jejunum and terminal ileum both before (control, –Dex) and after a 4-day treatment with dexamethasone (+Dex) are shown. Values are arithmetic means ± SE; n = 3–7. *Statistically significant difference (P < 0.05) between +Dex and –Dex.

View larger version (193K): [in this window] [in a new window]   Fig. 2. Localized intestinal expression of SGK1 mRNA. In situ hybridization of SGK1 mRNA abundance in cryostat sections of the adult mouse gut is shown. A and B: overview of SGK-1 mRNA expression in the small intestine of untreated (A) and Dex-treated (B) mice. Dex strongly increased SGK-1 expression in the small intestinal epithelium. C and D: High-magnification photomicrographs of A and B. Scale bars = 100 µm.

SGLT1 activity. To elucidate the in vivo significance of SGK1-dependent regulation of SGLT1, proximal and distal segments of the jejunum from sgk1^–/– and sgk1^+/+ mice were mounted into mini-Ussing chambers, and electrogenic glucose transport was determined using electrophysiological analysis. In the absence of luminal substrates, the V_t of proximal jejunal segments (V_t,p) amounted to –1.6 ± 0.5 mV (n = 9) in untreated sgk1^–/– mice and to –1.2 ± 0.3 mV (n = 9) in untreated sgk1^+/+ mice. The R_t of proximal jejunal segments (R_t,p) approached 31.0 ± 6.4 ·cm² (n = 9) in sgk1^–/– mice and 42.8 ± 7.7 ·cm² (n = 9) in sgk1^+/+ mice. Neither V_t nor R_t were significantly different between sgk1^–/– and sgk1^+/+ mice.

The partial isosmotic replacement of mannitol by glucose created a lumen-negative shift of the V_t,p (V_g,p) without significantly altering the R_t,p. No significant difference was observed between V_t,p and V_g,p in sgk1^–/– and sgk1^+/+ mice (Table 1). V_g,p and R_t,p allowed the calculation of the glucose-induced current (I_g) of proximal jejunal segments (I_g,p), which was again not significantly different between sgk1^–/– and sgk1^+/+ mice (Fig. 3).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Glucose-induced current (Ig) in the proximal (Ig,p) and distal (Ig,d) jejunum. Glucose-induced alterations of transepithelial voltages (Vg) and Ig in proximal and distal segments of jejunal tissue from SGK1 knockout (ko) mice (sgk1–/– mice) and their wild-type (wt) littermates (sgk1+/+ mice) without treatment (–Dex) or after a 4-day treatment with Dex (+Dex; 10 µg·g body wt–1·day–1) are shown. A: original tracings from typical experiments illustrating the effect of 10 mM glucose (addition indicated by arrows) on the transepithelial potential difference in untreated (top) and Dex-treated (bottom) sgk1+/+ (left) and sgk1–/– (right) mice. B: values are arithmetic means ± SE (n = 8–9) of Ig,p [change in short-circuit current (Isc)] from sgk1+/+ (open bars) and sgk1–/– (filled bars) mice treated with Dex (+Dex) or left untreated (–Dex). *Statistically significant difference (P < 0.05) between +Dex and –Dex; #statistically significant difference (P < 0.05) between the current in sgk1+/+ and sgk1–/– mice. C: Ig,d from sgk1+/+ and sgk1–/– mice without treatment or after a 4-day treatment with Dex (10 µg·g body wt–1·day–1). Values are arithmetic means ± SE (n = 12–13) of Ig,d from sgk1+/+ mice (open bars) and sgk1–/– mice (filled bars) treated with Dex (+Dex) or left untreated (–Dex). *Statistically significant difference (P < 0.05) between +Dex and –Dex; #statistically significant difference (P < 0.05) between sgk1+/+ and sgk1–/– mice.

In conclusion, I_g was similar in untreated sgk1^–/– and sgk1^+/+ mice but was sensitive to the stimulatory effect of dexamethasone only in sgk1^+/+ mice.

Similar observations were made in distal jejunal segments. The V_t of distal jejunal segments (V_t,d) in the absence of luminal glucose amounted to –0.85 ± 0.17 mV (n = 9) in untreated sgk1^–/– mice and to –0.79 ± 0.14 mV (n = 11) in untreated sgk1^+/+ mice. The R_t of distal jejunal segments (R_t,d) was 25.5 ± 2.6 ·cm² (n = 9) in sgk1^–/– mice and 25.5 ± 2.1 ·cm² (n = 11) in sgk1^+/+ mice. As in proximal jejunal segments, neither V_t,d nor R_t,d were significantly different between sgk1^–/– and sgk1^+/+ mice.

The shift of the V_t,d after partial isosmotic replacement of mannitol by glucose (V_g,d) was again not significantly different between untreated sgk1^–/– mice and untreated sgk1^+/+ mice (Table 1). V_g,d and R_t,d allowed the calculation of the I_g in distal jejunal segments (I_g,d), which was again not significantly different between sgk1^–/– and sgk1^+/+ mice (Fig. 3).

Treatment of the mice with dexamethasone did not significantly modify basal V_t,d in sgk1^–/– mice (–1.38 ± 0.28 mV, n = 12) or sgk1^+/+ mice (–1.01 ± 0.21 mV, n = 13). Similarly, R_t,d was not significantly altered by dexamethasone treatment (32.4 ± 4.3 ·cm² in sgk1^–/– mice and 26.4 ± 2.3 ·cm² in sgk1^+/+ mice). However, I_g,d was significantly enhanced after treatment of sgk1^+/+ mice with dexamethasone but remained without any effect in sgk1^–/– mice (Fig. 3). The values for I_g,d after dexamethasone treatment were significantly larger in sgk1^+/+ mice than in sgk1^–/– mice (Table 1).

The stimulation of I_g was paralleled by increases of SGLT1 protein abundance in brush-border membranes (Fig. 4). Treatment with dexamethasone led to a variable but significant increase of SGLT1 protein abundance in sgk1^+/+ mice. The SGLT1 protein abundance was not significantly altered by dexamethasone treatment in sgk1^–/– mice (Fig. 4).

View larger version (27K): [in this window] [in a new window]   Fig. 4. Sodium-coupled glucose transporter 1 (SGLT1) protein abundance in the intestinal brush border. A: original Western blot of SGLT1 protein abundance in the intestinal brush-border membrane in tissue from WT mice and SGK1 KO mice without treatment or after a 4-day treatment with Dex (10 µg·g body wt–1·day–1). B: values are arithmetic means ± SE (n = 4–5) of protein density [in arbitrary units (a.u.)] in intestinal brush-border membrane of WT and SGK1 KO mice without treatment or after a 4-day treatment with Dex. *Statistically significant difference (P < 0.05) between Dex-treated and untreated mice; #statistically significant difference (P < 0.05) between sgk1+/+ and sgk1–/– mice.

NHE3 activity. Measurements of pH_i were utilized to determine NHE3 activity. The initial pH_i was similar in untreated sgk1^+/+ mice (7.18 ± 0.01, n = 6) and sgk1^–/– mice (7.16 ± 0.01, n = 5) and was not significantly affected by treatment with dexamethasone of sgk1^+/+ mice (7.15 ± 0.01, n = 7) and sgk1^–/– mice (7.16 ± 0.01, n = 7).

Ammonium pulses were utilized to load the cells with H⁺ (52). The superfusion with 20 mM NH₄Cl and simultaneous removal of Na⁺ (replacement with NMDG⁺) was followed by slight alkalinization (Fig. 5). The subsequent replacement of NH₄Cl with NMDG⁺ Cl^– in the continued absence of Na⁺ led to a sharp acidification due to exit of NH3 and retention of H⁺ within the cells. The acidification allowed the calculation of an apparent buffer capacity (see METHODS) that was similar in untreated sgk1^+/+ mice (60.0 ± 2.3 mM/pH, n = 6) and sgk1^–/– mice (59.6 ± 3.4 mM/pH, n = 5) and was not significantly affected by treatment with dexamethasone in either sgk1^+/+ mice (56.5 ± 2.0 mM/pH, n = 7) or sgk1^–/– mice (60.6 ± 2.4 mM/pH, n = 7). pH recovery in the absence of Na⁺ was minimal in untreated or treated sgk1^+/+ and sgk1^–/– mice (Table 2) but markedly accelerated by subsequent replacement of NMDG⁺ with Na⁺ (Fig. 5). The Na⁺-dependent realkalinization was not significantly different in untreated sgk1^+/+ and sgk1^–/– mice (Fig. 5). Treatment with dexamethasone led to a significant increase of Na⁺-dependent realkalinization in sgk1^+/+ and sgk1^–/– mice (Table 2), an effect significantly larger in sgk1^+/+ than sgk1^–/– mice (Fig. 5).

View larger version (13K): [in this window] [in a new window]   Fig. 5. pH recovery in the ileum after an ammonium pulse. Alterations of cytosolic pH (pH) in the ileum after an ammonium pulse are shown. To load the cells with H+, 20 mM NH4Cl was added and Na+ was removed (replaced by NMDG) in the first step (see bars below each original tracing), NH4Cl was removed in the second step, Na+ was added in the third step, and nigericin (Nig) was applied in the fourth step to calibrate each individual experiment. A: original tracings illustrating alterations of pH in typical experiments in untreated (top) and Dex-treated (bottom) sgk1+/+ mice (left) and sgk1–/– mice (right). B: values are arithmetic means ± SE (n = 5–7 animals, 67–116 cells) of pH in the ileum from sgk1+/+ mice (open bars) and sgk1–/– mice (filled bars) treated with Dex (+Dex) or left untreated (–Dex). Left: in the absence of inhibitors; right: in the presence of S-3226. Inset: S-3226-sensitive pH recovery under the respective conditions. *Statistically significant difference (P < 0.05) between +Dex and –Dex; #statistically significant difference (P < 0.05) between sgk1+/+ and sgk1–/– mice.

The stimulation of NHE3 activity was paralleled by increases of NHE3 protein abundance in brush-border membranes (Fig. 6). Treatment with dexamethasone significantly increased the NHE protein abundance in sgk1^+/+ mice but not in sgk1^–/– mice. Accordingly, the NHE protein abundance was significantly higher in dexamethasone-treated sgk1^+/+ mice than in dexamethasone-treated sgk1^–/– mice (Fig. 6).

View larger version (37K): [in this window] [in a new window]   Fig. 6. Na+/H+ exchanger 3 (NHE3) protein abundance in the intestinal brush border. A: original Western blot of NHE3 protein abundance (in a.u.) in the intestinal brush border membrane from WT and SGK1 KO mice without treatment or after a 4-day treatment with Dex (10 µg·g body wt–1·day–1). B: values are arithmetic means ± SE (n = 4–5) of NHE3 protein density in the intestinal brush-border membrane of WT and SGK1 KO mice without treatment or after a 4-day treatment with Dex. *Statistically significant difference (P < 0.05) between Dex-treated and untreated mice; #statistically significant difference (P < 0.05) between sgk1+/+ and sgk1–/– mice.

	DISCUSSION

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The present observations provide the first demonstration of an intestinal phenotype of the SGK1 knockout mouse. Moreover, it is demonstrated for the first time that lack of SGK1 indeed affects the regulation of organ function by glucocorticoids. Earlier observations have indicated that glucocorticoids upregulate the intestinal transport of nutrients (31) and stimulate the activity of the apical Na⁺/H⁺ exchanger (NHE3) (33, 43, 63).

According to in vitro studies, SGK1 enhances the cell membrane protein abundance of various ion channels and carriers (41), including SGLT1 (19) and NHE3 (65, 66). Thus it appeared likely that the effects of glucocorticoids were mediated by SGK1. If so, the effect of the glucocorticoids should have been absent in gene-targeted mice lacking functional SGK1 (sgk1^–/– mice). This is indeed true for glucocorticoid regulation of SGLT1.

The present experiments revealed that the lack of SGK1 does not affect intestinal Na⁺-coupled glucose transport in untreated animals but abrogates the stimulating effect of glucocorticoids on glucose transport. The apparently normal glucose absorption in the untreated sgk1^–/– mice raises the question of which mechanism could replace SGK1 in the absence of glucocorticoid stimulation. Candidate kinases possibly replacing SGK1 include the isoforms SGK2 and SGK3, which have been cloned by homology screening (36) and share the ability of SGK1 to enhance the protein abundance and/or activity of several channels and transporters (41). Experiments in Xenopus oocytes indeed disclosed the ability of SGK3 to upregulate SGLT1 (19). Moreover, in those experiments, the related protein kinase B has been shown to upregulate SGLT1 activity (19). Protein kinase B has previously been shown to enhance the abundance of several glucose transporters in the cell membrane (28, 38, 57). In contrast to SGK1, there is no published evidence for transcriptional regulation of SGK2 and SGK3 by glucocorticoids (40). Thus it appears that SGK2, SGK3, and protein kinase B are constitutively expressed. All three SGK isoforms and protein kinase B require activation by insulin and growth factors including IGF-I (40). Thus all those kinases may participate in the known stimulating effect of IGF on intestinal nutrient uptake (4, 16, 39). Presently available evidence indicates that the transcriptional upregulation of SGK1 by glucocorticoids and mineralocorticoids might serve to sensitize the tissues for the effects of insulin and growth factors. Thus dexamethasone, through upregulation of SGK1, could allow the enhanced stimulation of intestinal nutrient uptake by IGF-I, an effect lacking in the SGK1 knockout mouse.

Glucocorticoids increase the S-3226-sensitive portion of Na⁺-dependent pH recovery, consistent with the earlier observation that glucocorticoids upregulate NHE3 (63). The glucocorticoid regulation of NHE3 is only partially dependent on SGK1. Glucocorticoids increase NHE3 abundance in part by stimulation of expression (14, 32), an effect presumably not requiring the participation of SGK1.

SGK1 rather affects the trafficking of NHE3 to the cell membrane, an effect requiring the participation of NHE-regulating factor 2 (65, 66). Other transport proteins, including SGLT1 (19), are regulated by SGK1 through phosphorylation of the ubiquitin ligase Nedd4-2, which decreases the affinity of the enzyme to its target proteins (1, 18, 55, 56). Both enhanced transcription and membrane trafficking or delayed retrieval by impaired ubiquitination would enhance protein abundance in the plasma membrane. Indeed, Western blots have demonstrated increases of protein abundance after dexamethasone treatment in sgk1^+/+ mice but not in sgk1^–/– mice. The limited accuracy of Western blots precludes, however, the quantification of minor changes in protein abundance. SGK1 may further be effective through direct phosphorylation of channels or carrier proteins (41). Irrespective of the underlying mechanisms, the present observations clearly demonstrate that the effect of dexamethasone on intestinal transport requires the participation of SGK1.

Similar to the regulation of SGLT1, the regulation of NHE3 by glucocorticoids might lead to the sensitization against the effects of insulin (34). By upregulation of SGK1, glucocorticoids could sensitize the tissue for the stimulation of NHE3 activity.

The role of SGK1 in the glucocorticoid regulation of intestinal SGLT1 and NHE3 is thus similar to the role of SGK1 in the mineralocorticoid regulation of renal Na⁺ and K⁺ excretion. SGK1 transcription is upregulated by mineralocorticoids (15, 46, 54). In Xenopus oocytes expressing the ,,-subunits of ENaC, coexpression of SGK1 results in a marked upregulation of Na⁺ channel activity (7, 12, 15, 42, 44, 58). Urinary Na⁺ excretion is, however, seemingly normal in sgk1^–/– mice (64), contrasting with the severe phenotype of mice lacking functional -ENaC (29, 30), -ENaC (45), -ENaC (8), or the mineralocorticoid receptor (9). The defective regulation of renal Na⁺ excretion becomes apparent only after exposure to a salt-deficient diet, which unmasks the limited ability of the sgk1^–/– mice to decrease their urinary Na⁺ output (64).

In conclusion, in the absence of exogenous glucocorticoids, little differences are observed between sgk1^–/– and sgk1^+/+ mice in the activities of intestinal SGLT1 and NHE3. Thus basal activities of those intestinal transport proteins do not depend on SGK1. However, the glucocorticoid stimulation of SGLT1 is fully, and the glucocorticoid stimulation of NHE3 partially, dependent on SGK1. Thus regulation of intestinal transport by glucocorticoids involves both SGK1-dependent and -independent mechanisms.

	GRANTS

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This work was supported by grants from DFG and BMBF (to F. Lang) as well as Fortüne (to B. Friedrich).

	ACKNOWLEDGMENTS

 
The authors gratefully acknowledge the expert technical assistance by Andrea Janessa and the meticulous preparation of the manuscript by Lejla Subasic. The specific NHE3 inhibitor S-3226 was a kind gift from H. J. Lang and A. Busch from Aventis.

	FOOTNOTES

 

Address for reprint requests and other correspondence: F. Lang, Dept. of Physiology, Univ. of Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany (e-mail: florian.lang{at}uni-tuebingen.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^* F. Grahammer and G. Henke contributed equally to this work.

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