AJP - GI Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 292: G1089-G1098, 2007. First published January 11, 2007; doi:10.1152/ajpgi.00088.2006
0193-1857/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/4/G1089    most recent
00088.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schäfer, C.
Right arrow Articles by Schliess, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schäfer, C.
Right arrow Articles by Schliess, F.

HORMONES AND SIGNALING

Osmotic regulation of betaine homocysteine-S-methyltransferase expression in H4IIE rat hepatoma cells

Christine Schäfer,1,* Lars Hoffmann,2,* Katrin Heldt,2 Mohammad Reza Lornejad-Schäfer,1 Gernot Brauers,1 Thor Gehrmann,1 Timothy A. Garrow,3 Dieter Häussinger,1 Ertan Mayatepek,2 Bernd C. Schwahn,2 and Freimut Schliess1

1Clinic for Gastroenterology, Hepatology, and Infectiology and 2Heinrich-Heine-University, Clinic for General Paediatrics, Düsseldorf, Germany; and 3Department of Food Science and Human Nutrition, University of Illinois, Urbana-Champaign, Illinois

Submitted 24 February 2006 ; accepted in final form 28 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell hydration changes critically affect liver metabolism and gene expression. In the course of gene expression studies using nylon cDNA-arrays we found that hyperosmolarity (405 mosmol/l) suppressed the betaine-homocysteine methyltransferase (Bhmt) mRNA expression in H4IIE rat hepatoma cells. This was confirmed by Northern blot and real-time quantitative RT-PCR analysis, which in addition unraveled a pronounced induction of Bhmt mRNA expression by hypoosmotic (205 mosmol/l) swelling. Osmotic regulation of Bhmt mRNA expression was largely paralleled at the levels of Bhmt protein and enzymatic activity. Like hyperosmotic NaCl, hyperosmotic raffinose but not hyperosmotic urea suppressed Bhmt mRNA expression, suggesting that cell shrinkage rather than increased ionic strength or hyperosmolarity per se is the trigger. Hypoosmolarity increased the expression of a reporter gene driven by the entire human BHMT promoter, whereas destabilization of BHMT mRNA was observed under hyperosmotic conditions. Osmosensitivity of Bhmt mRNA expression was impaired by inhibitors of tyrosine kinases and cyclic nucleotide-dependent kinases. The osmotic regulation of BHMT may be part of a cell volume-regulatory response and additionally lead to metabolic alterations that depend on the availability of betaine-derived methyl groups.

cell volume; osmolytes; gene expression; methylation; liver

BETAINE (TRIMETHYLGLYCINE) is a compatible organic osmolyte that is available for cells by either choline oxidase-catalyzed synthesis (43) or concentrative uptake by the betaine/GABA transport system BGT-1 (4). The intracellular accumulation of betaine protects cells from different kind of stress including long-lasting dehydration (4), ischemia-reperfusion (41), and heat shock (33). Betaine has been considered a "chemical chaperone" because it prevents protein denaturation under adverse conditions (17), a property that likely contributes to the cytoprotective effects of this osmolyte.

Betaine is metabolically eliminated by betaine homocysteine S-methyltransferase (BHMT, EC 2.1.1.5 [EC] ), an enzyme that catalyzes a methyl group transfer from betaine to homocysteine to form dimethylglycine (DMG) and methionine (43). BHMT is expressed at high levels in the human liver and kidney and the rat liver (34), and present knowledge suggests that its activity is largely regulated at the expression level (24). Remethylation of homocysteine through BHMT competes with the alternative methylfolate-dependent remethylation, catalyzed by methionine synthase. BHMT activity becomes essential when folate-dependent remethylation is impaired because of nutritional deficiencies (folate or cobalamin deficiency) or genetic defects of 5,10-methylenetetrahydrofolate reductase, cystathionine-beta-synthase, or cobalamin processing, respectively (30–32). Betaine supplementation is effective in the treatment of human homocystinuria (40). In mouse models of homocystinuria due to methylenetetrahydrofolate reductase or cystathionine-beta-synthase deficiency, hyperhomocysteinemia and betaine depletion were associated with fatty liver, which was preventable by betaine supplementation (30, 32). Bhmt gene expression is severely decreased in rat liver cirrhosis (9), providing more evidence for an association between disrupted betaine metabolism and liver disease.

The cellular hydration state is dynamic despite the presence of cell volume regulatory mechanisms that prevent excessive cell swelling or shrinkage. Cell hydration changes induced by either anisoosmolarity or under isoosmotic conditions by hormones, substrates, or oxidative stress contribute to the regulation of cellular metabolism and gene expression (12, 15, 28). Cell swelling, in general, supports anabolic metabolism and cell protection, whereas cell shrinkage favors catabolism and insulin resistance and sensitizes cells to different kinds of stress (12). The metabolism of betaine under anisoosmotic conditions has not yet been thoroughly studied. Only recently, high salt intake of NaCl was shown in vivo to decrease Bhmt activity in the liver and kidney but not pancreas in the guinea pig (7).

Cells and tissues exposed to anisoosmolarity are frequently used as experimental paradigm to study the dependence of metabolism and gene expression on cell hydration. Here, the osmosensitivity of Bhmt was studied in H4IIE rat hepatoma cells and mouse hepatocytes. The results demonstrated a clear osmosensitive regulation of Bhmt. The hyperosmotic downregulation of Bhmt expression was related to cell shrinkage rather than to an elevation of ionic strength or osmolarity per se.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Cell culture media and FCS were from GIBCO Life Technologies (Gaithersburg, MD). The STET-lysis buffer (pH 8) used for the metabolite analysis was from Fluka (Buchs, Switzerland). Actinomycin D, EDTA, sodium vanadate, Nonidet (NP-40), Triton X-100, dithiothreitol, DMG, and betaine anhydrous were obtained from Sigma (Munich, Germany). Genistein, daidzein, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine·2HCl (H7), N-{2-[(p-bromocinnamyl)amino]ethyl}-5-isoquinolinesulfonamide·2HCl (H89), wortmannin, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), 2'-amino-3'-methoxyflavone (PD098059), and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4'-pyridyl)1H-imidazole (SB203580) were from Calbiochem (Darmstadt, Germany), and 1,9-pyrazoloanthrone (SP600125) was from BioMol (Hamburg, Germany). Gö6850 was a gift from Gödecke (Freiburg, Germany). HPLC grade methanol and water, ammonia solution (25%), and all other chemicals were from Merck (Darmstadt, Germany). d6-DMG was from CDN Isotopes (Quebec, Canada); d11-betaine was purchased from Cambridge Isotope Laboratories (Andover). Protein content was determined by using the BCA protein assay kit (Pierce, Rockford, IL). Polyclonal antibodies were raised in rabbits against highly purified human BHMT (21). The anti-GAPDH antibody was from Biodesign (Cologne, Germany). Densitometric analysis was performed with the E.A.S.Y RH system (Herolab, Wiesloch, Germany) and Scion Image software.

Plasmids. The isolation of genomic DNA encoding exons 1 and 2 of the human BHMT gene and a large segment of 5'-flanking sequence and the subsequent cloning of this DNA fragment into vector pBluescript II KS (Stratagene, La Jolla, CA) to form plasmid pEP8 have previously been described (23). The DNA comprising region –3179 to –28 of the 5' untranslated region of the human BHMT gene was isolated from pEP8 by the following steps. First, a SalI digest of pEP8 gave an ~10-kb fragment that was gel purified and further digested with KpnI. The resulting ~3.2-kb product was then gel purified and ligated into pGL3-Basic (Promega, Madison, WI), which had been linearized with KpnI and XhoI. The ligase reaction product was then transformed into Escherichia coli strain XL1-Blue (Stratagene, La Jolla, CA), and the resulting construct was verified by sequencing. This promoter construct is referred to as the KpnI construct.

The DNA comprising region –424 to –28, comprising what is referred to as the 400-nt construct, was generated from the KpnI construct as follows. First, the KpnI construct was digested with StuI, and, following a sufficient incubation period, the restriction enzyme was heat inactivated and the DNA ends were blunted by using T4 DNA polymerase (New England Biolabs, Ipswich, MA) following the manufacturer's instructions. The resulting fragment containing the pGL3-Basic vector plus the ~400-nt promoter sequence was gel purified, and its blunt ends were ligated together. The integrity of the 400-nt construct was verified by sequencing.

Cell culture and experimental treatment of the cells. H4IIE-C3 rat hepatoma cells (American Type Culture Collection CRL 1600) were maintained in Cluster six dishes (Greiner bio-one, Frickenhausen, Germany) containing DMEM-F-12 media supplemented with 5 mM glucose and 10% FCS. Hepatocytes were prepared from livers of male B57CL/6 mice fed ad libitum with a standard diet by a collagenase perfusion technique as described previously (26). Cells were grown at 37°C in 5% CO2. Hepatocytes and H4IIE cells were washed with Dulbecco's PBS, and the culture was continued in 1.5 ml of serum-free medium for an additional 15–24 h. Hyperosmolarity (405 mosmol/l) and hypoosmolarity (205 mosmol/l) were adjusted by dilution with medium of elevated NaCl concentration or without NaCl, respectively. In the normoosmotic control (305 mosmol/l), an identical volume of normoosmotic medium was added.

Custom nylon array hybridization. Custom nylon arrays (Atlas rat toxicology 1.2 arrays, Clontech Laboratories, Palo Alto, CA) were used according to the manufacturer's recommendations. Total RNA (5 µg) was reverse transcribed in the presence of [{alpha}-32P]dATP by using gene-specific primers. Unincorporated nucleotide was removed by two rounds of column chromatography (BioSpin 30, Bio-Rad, Hercules, CA). The array membranes were prehybridized for 30 min in ExpressHyb (Clontech, Mountain View, CA) and 100 µg/ml sheared salmon testes DNA (Eppendorf, Waterbury, NY). The probe was chemically denatured and membranes were hybridized at 68°C. The arrays were washed four times for 30 min each in 2x SSC and 1% SDS at 68°C, once with 0.1x SSC and 0.5% SDS for 30 min at 68°C, and then briefly rinsed in room temperature 2x SSC. After being washed, the membranes were exposed to a phosphoimager screen and were scanned with an Fuji FLA 8000-scanner (Raytest, Straubenhardt, Germany) with the following parameters: excitation 532 nm, emission filter 570 nm, resolution 10 µm, scan speed 200 mm/s, and photomultiplier voltage 100%.

Northern blot analysis of Bhmt mRNA expression. RNA samples (10 µg) were electrophoresed in 0.8% agarose-3% formaldehyde and then RNAs were blotted onto Hybond-N nylon membranes (Amersham Pharmacia Biotech Europe, Freiburg, Germany) with 20x SSC (3 M NaCl, 0.3 M sodium citrate). After being briefly rinsed with water and cross-linked (Hoefer UV-cross-linker 500; Hoefer, San Francisco, CA), the membranes were inspected under UV light to determine RNA integrity and the location of the 28S and 18S rRNA spots. Blots were then subjected to a 3-h prehybridization at 43°C in 50% deionized formamide in sodium phosphate buffer (0.26 M, pH 7.2) containing 0.25 M NaCl, 1 mM EDTA, 100 µg/ml salmon sperm DNA, and 7% SDS. Hybridization was performed in the same solution with ~106 counts·min–1·ml–1 [{alpha}-32P]dCTP cDNA probe (Rediprime labeling kit; Amersham Pharmacia Biotech, Little Chalfont, UK). Membranes were washed three times in 2x SSC-0.1% SDS for 15 min, twice in sodium phosphate buffer (25 mM, pH 7.2)-EDTA (1 mM)-0.1% SDS for 10 min, and twice in sodium phosphate buffer for 10 min at 53°C. Blots were then exposed to Kodak AR X-Omat film (Eastman Kodak, Rochester, NY) at –70°C with intensifying screens.

The Bhmt cDNA fragment used as a probe was synthesized from 1 µg total RNA through the first strand cDNA kit for RT-PCR (AMV) from Roche Molecular Biochemicals (Mannheim, Germany) with oligo(dT) primers. The following primers were used to amplify a 430-bp fragment according to the rat BHMT nucleotide sequence (GenBank accession no. AF038870): 5'-TTGCAGGAGGTGTGAGTCAG-3' (upstream primer) and 5'-AATCCCTGTTTGCCACAGTC-3' (downstream primer). The primers were chosen by using the Primer3 program (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) and were synthesized by MWG Biotech (Ebersberg, Germany).

The PCRs were performed in 50 µl of the following reaction mixture: 1x reaction buffer, 2.5 mM MgCl2, 0.2 mM dNTP mix, 0.2 pM each primer, 1.25 units Goldstar DNA-polymerase (Eurogentec, Seraing, Belgium), and 100 ng cDNA. The reaction was performed in a PE Biosystems GeneAmp PCR System 2400. The following program was used: 5 min at 95°C; 40 cycles (40 s at 95°C; 50 s at 60°C; and 1 min at 72°C); and 7 min at 72°C. The PCR products were separated by agarose gel electrophoresis and eluted by using the Qiaquick Gel extraction kit (Qiagen, Hilden, Germany). Sequence analysis confirmed the specificity of the PCR amplification and was performed by the Biologisch-Medizinisches Forschungszentrum (Heinrich-Heine-University, Düsseldorf, Germany).

RT-PCR-analysis of Bhmt mRNA expression. To determine the expression of Bhmt mRNA, we used real-time PCR. Cyclooxygenase (Cox) IV mRNA expression was unaffected by our experimental design and was used as a reference. One sense and two antisense primers were designed for each transcript with the help of FastPCR program (R. Kalendar, Institute of Biotechnology/Helsinki; http://www.biocenter.helsinki.fi/bi/Programs/fastpcr.htm) with a melting temperature = 58–63°C and a GC content of 50–60% (Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Primers used for quantitative real-time PCR of the Bhmt and Cox IV rat genes

 
We isolated total RNA from H4IIE cells using the RNeasy mini kit (Qiagen, Hilden, Germany). Quality and quantity of total RNA were determined spectrometrically. RT and PCR were done in a single-step procedure using the QuantiTect SYBR Green RT-PCR kit from Qiagen (Hilden, Germany) with 150 ng of total RNA as a template. Real-time PCR was performed on the Applied Biosystems 7700 sequence-detection system in MicroAmp 96-well optical reaction plates capped with MicroAmp optical caps (Applied Biosystems, Foster City, CA). The thermal protocol was subdivided into three stages: stage 1, 30 min at 50°C; stage 2, 15 min at 95°C; and stage 3, 15 s at 95°C, 60 s at 60°C, and 60 s at 72°C for 40 cycles. The specificity of amplification was determined after the last cycle of the PCR reaction by dissociation curve analysis. Each reaction showed a single amplification product.

Real-time PCR data were plotted as fluorescence signal (Rn) vs. cycle number. Rn was calculated by using the equation {theta}Rn = (Rn+) – (Rn–), where Rn+ is the fluorescence signal of the product at any given time and Rn– is the fluorescence signal of the baseline emission during cycles 6–15. An arbitrary threshold was set at the midpoint of log Rn vs. cycle number plot. The cycle threshold (Ct) value is defined as the cycle number at which the Rn crosses this line. The relative difference in target cDNA concentration was determined by {Delta}Conc = 2 exp(Ct A – Ct B), where A is the experimental sample and B represents the respective control at isoosmolar conditions. Bhmt mRNA/cDNA concentration was normalized for the concentrations of Cox IV mRNA/cDNA in the same sample.

Western blot analysis of Bhmt expression. At the end of the experimental treatment, medium was removed from the culture and cells were immediately lysed at 4°C by use of 10 mM Tris·HCl buffer (pH 7.4) containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, and 1 tablet per 50 ml of protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The homogenized lysates were centrifuged at 20,000 g at 4°C for 10 min. To perform SDS PAGE and Western blot analysis, the supernatant was added to an identical volume of 2x gel loading buffer containing 200 mM DTT (pH 6.8). After being heated to 95°C for 5 min, proteins were subjected to SDS-PAGE (60 µg protein/lane). Following electrophoresis, gels were equilibrated with transfer buffer (39 mM glycine, 48 mM Tris·HCl, 0.03% SDS, and 20% methanol). Proteins were transferred to nitrocellulose membranes using a semi-dry transfer apparatus (Pharmacia, Freiburg, Germany). Blots were blocked overnight in 5% BSA solubilized in 20 mM Tris·HCl (pH 7.5) containing 150 mM NaCl and 0.1% Tween 20 and then incubated for 3–4 h with anti-BHMT diluted 1:3,000. Following a wash and incubation for 2 h with horseradish peroxidase-coupled anti-rabbit-IgG antibody (1:10,000–1:20,000, 4°C), the blots were washed again and developed by enhanced chemiluminescent detection (Amersham Biosciences Europe, Freiburg, Germany).

Transfection procedure and reporter gene assay of BHMT promoter activity. For magnetofection (MF), H4IIE cells were seeded 1 day before MF in six-well plates (200,000/well) and grown in 2 ml DMEM-F-12 containing 10% FCS. Before MF, at a confluency of about 80%, medium was removed and 1.5 ml of serum-free DMEM-F-12 was added. For preparation of the MF mixture, the desired amount of pDNA (2 µg DNA/well, ratio 10:1 experimental vector-control plasmid) was mixed with 500 µl of DMEM, followed by addition of PolyMAG (Chemicell, Berlin) and gentle mixing by pipetting up and down five times. The PolyMAG to pDNA ratio was 1:1. For controls, PolyMAG alone was added to 500 µl of DMEM. After an incubation at room temperature for 20 min, DNA/PolyMAG solution was added to the cells. For MF, six-well plates were placed in a magnetic field provided by three Nd-Fe-B magnets, each 25 mm in diameter (IBS Magnete, Berlin, Germany), for 30 min at room temperature. Medium was removed after transfection and fresh DMEM-F-12 with an ambient osmolarity of 205, 305, or 405 mosmol/l, respectively, was added. Osmolarity was adjusted by different concentrations of NaCl.

Twenty-four hours after MF, luciferase expression was measured with the Dual-Luciferase Reporter System (Promega). Cells were thoroughly washed once with PBS, and 100 µl of passive lysis buffer (Promega) were added. Cells were lysed for 15 min at room temperature, and 20 µl of each sample were measured in an Ascent Luminometer (Labsystems, Thermo, Germany). For quantitation of luciferase activity, 100 µl of luciferase assay puffer were injected to each sample, and light emission over 10 s was measured. After a 2-s lag time, 100 µl of stop and glo reagent (Promega) were added to each sample to measure Renilla luciferase activity for another 10 s. The background of each well was measured before injection and subtracted from the reported values. Each experiment was done in triplicate and measured twice. Means of three independent experiments were used to calculate means and SE.

Determination of Bhmt activity. Protein lysates were analyzed for Bhmt activity as described previously (10).

Determination of betaine and DMG. Betaine and DMG in cell lysates and medium were measured by HPLC-tandem mass spectrometry. A Waters 2795 Alliance HPLC system (Waters, Milford, UK), equipped with a thermostated autosampler, a column oven, and a degasser, was used for solvent delivery and sample introduction. Lysate samples deproteinized with TCA (10%) were placed in a cooled sample tray, and 30 µl were injected into a Agilent Zorbax 300 SB-C8 column (Agilent, Waldbronn, Germany). DMG and betaine were eluted with solution A (0.005% ammonia in water, pH 9.0) and solution B (0.65 M acetic acid, pH 2.0) at a flow rate of 0.8 ml/min with the following steps: 0–0.5 min, 100% A; 0.5–3.0 min 100% B; and 3.0–10.0 min 100% A. One-half of the eluate was delivered into a Quattro micro API tandem mass spectrometer (Micromass, Cambridge, UK) with a ESI probe in the positive ion mode. The injection interval was 12 min. Nitrogen was used as a drying gas at a flow rate of 650 l/h. The collision energy using argon as a collision gas was 12 eV for DMG and DMG d6 and 16 eV for betaine and d11-betaine. The declustering potential was 18 V for DMG and 31 V for betaine, and the ion source temperature was 100°C. Compounds were detected in the multiple reaction monitoring mode with the following transitions: DMG, mass-to-charge ratio (m/z) 103.8 -> 57.7; d6-DMG, m/z 109.8 -> 63.6; betaine, m/z 117.9 -> 58.5; d11-betaine, m/z 128.9 -> 67.6. Data were acquired and analyzed using MassLynx NT v4.0 software (GV Instruments, Manchester, UK). We determined endogenous DMG and betaine concentrations in pooled human plasma by HPLC as described before (16) and found 3.0 µmol/l DMG and 29.2 µmol/l betaine. Plasma was spiked with DMG and betaine to produce standards in the range of 3.0–120.0 µmol/l for DMG and 29.2–150 µmol/l for betaine. Another standard series was obtained by spiking lysates of H4IIE cells. These standards were used to assess linearity and sensitivity of our method. Concentrations of samples were calculated from peak-area ratios of DMG to d6-DMG and of betaine to d11-betaine.

Analysis of results. Results are from three independent experiments in triplicate and are expressed as means ± SE. Results were compared using one-way or two-way ANOVA, as appropriate, followed by Bonferroni's multiple-comparison test or single t-tests. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Bhmt mRNA steady-state concentration is sensitive to ambient osmolarity. To study osmosensitivity of gene expression, H4IIE rat hepatoma cells were exposed to normoosmolarity (305 mosmol/l) or hyperosmolarity (405 mosmol/l) adjusted by addition of 50 mmol/l NaCl for 8 h. RNA was isolated and used to prepare probes for the hybridization of commercially available cDNA arrays. These studies unraveled a suppression of Bhmt mRNA expression by hyperosmolarity (Fig. 1).


Figure 1
View larger version (48K):
[in this window]
[in a new window]

  		Fig. 1. Hyperosmotic suppression of betaine homocysteine S-methyltransferase (Bhmt) mRNA expression. H4IIE cells were exposed for 8 h to normoosmotic (305 mosmol/l) or hyperosmotic (405 mosmol/l) medium. At the end of the experimental treatment, cell lysates were collected for RNA preparation. Nylon cDNA arrays were probed with 32P-labeled cDNAs prepared from the mRNA samples. Arrows indicate the signals representing Bhmt mRNA expression (position C07j). Visual inspection indicated that hyperosmolarity decreases Bhmt mRNA expression. For comparison, the signals representing expression of the ribosomal proteins S19 and L15 are also indicated (positions C13m and C13l, respectively). Expression of S19 and L15 remain largely unaffected by hyperosmolarity. A representative of 5 independent experiments is shown.

 
We further studied the osmosensitivity of Bhmt mRNA expression by Northern blot analysis. As shown in Fig. 2A, hypoosmolarity (280–205 mosmol/l) adjusted by removal of NaCl gradually increased Bhmt mRNA expression up to 2.30 ± 0.10-fold. Already slight hyperosmolarity (355 mosmol/l) effectively suppressed Bhmt mRNA toward a 0.47 ± 0.10-fold expression (one-way ANOVA for osmolarity: P < 0.0001).


Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 2. Osmosensitivity of Bhmt mRNA expression. Cells were exposed to the normoosmotic (305 mosmol/l) control medium or to anisoosmotic media for the time periods indicated. RNA was prepared and analyzed for the presence of Bhmt mRNA by either Northern blot or quantitative RT-PCR. A: osmolarity dependence in H4IIE cells. For densitometric quantification of the Northern blots, normalized Bhmt mRNA expression (ratio Bhmt mRNA to ribosomal 18S RNA) under the normoosmotic control condition was taken as onefold expression. B: time course in H4IIE cells. Means and SEs of 3 independent experiments are shown. Results were normalized to the expression of cyclooxygenase IV (Cox IV) and are shown relative to the respective isoosmotic condition. *Bhmt mRNA expression significantly different from that found under the normoosmotic control condition. C: osmotic regulation of Bhmt mRNA expression in cultured mouse hepatocytes (n = 3). Results were analyzed by RT-PCR as described in B.

 
We confirmed the osmosensitive Bhmt mRNA expression by real-time quantitative RT-PCR (Fig. 2B). With this method, we detected a 4.32 ± 0.66-fold upregulation of Bhmt by hypoosmolarity (205 mosmol/l) compared with normoosmolarity, whereas hyperosmolarity downregulated the relative expression to 0.09 ± 0.01, i.e., by more than 90% at the 24-h time point. Osmotic modulation of Bhmt mRNA expression was easily detected after only 4 h of treatment, with the maximum responses obtained at 8 and 16 h for the hyperosmotic and hypoosmotic conditions, respectively (Fig. 2B). The osmosensitivity of Bhmt mRNA expression was also observed in cultured mouse hepatocytes (Fig. 2C).

Pharmacological characterization of the osmotic modulation of Bhmt mRNA expression. Signals mediating the osmosensitivity of Bhmt mRNA expression were analyzed by pharmacological means (Fig. 3). Genistein, a tyrosine kinase inhibitor (1), but not daidzein, its inactive analog, impaired the hypoosmotic stimulation of Bhmt mRNA expression but was largely without effect on hyperosmotic Bhmt mRNA suppression. H89, a PKA inhibitor (8), and the broad-spectrum kinase inhibitor H7, at a dose that was within the range of Ki values for PKA, PKG, and PKC (13), impaired the hyperosmotic suppression of Bhmt mRNA expression. H89, in addition, inhibited the hypoosmotic induction of Bhmt mRNA. On the other hand, Gö68960, a broad-specificity PKC inhibitor (20), had only minor effects, indicating that PKC is probably not involved. Also, the phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 (5, 6), at concentrations that blunted PI3K-dependent, insulin signaling in H4IIE cells (18), and the highly specific mammalian target of rapamycin (mTOR) inhibitor rapamycin (6a) at concentrations that blocked insulin signaling toward the mTOR downstream targets 4E-BP1 and p70 S6-kinase in H4IIE cells (18), affected the osmosensitivity of Bhmt mRNA expression only to a lesser degree.


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 3. Pharmacological characterization of the osmosensitivity of Bhmt mRNA expression. H4IIE cells were exposed for 8 h to ambient hypo-, normo-, or hyperosmolarity without further treatment or following a 20-min pretreatment with genistein (Gen, 50 µmol/l), daidzein (Dai, 50 µmol/l), H89 (10 µmol/l), H7 (20 µmol/l), Gö6850 (10 µmol/l), wortmannin (Wo, 500 nmol/l), LY294002 (LY, 20 µmol/l), rapamycin (Rapa, 500 nmol/l), SB220025 (SB, 20 µmol/l), SP600125 (SP, 50 µmol/l), or PD098059 (PD, 20 µmol/l). The respective inhibitors remained present during the entire experimental incubation. Samples were prepared and analyzed for relative Bhmt mRNA expression levels by RT-PCR as described in Fig. 2. *Bhmt mRNA expression differs significantly from the respective normoosmotic control condition; §Bhmt mRNA induction differs significantly from that found in absence of the inhibitor under the respective condition (n = 3).

 
Hypoosmolarity in H4IIE cells does not activate MAPKs, whereas hyperosmolarity activates ERK-1/ERK-2, JNK, and p38-type MAPK in these cells (29, 42). PD098059, SB220025, and SP600125 inhibit signaling via ERK-1/ERK-2, p38, and JNK, respectively (2, 3, 14) and were used to study a potential role of MAPKs in hyperosmotic Bhmt mRNA suppression. Although these compounds are effective in H4IIE cells (18, 19, 29), they do not affect the hyperosmotic suppression of Bhmt mRNA expression.

The data point to an involvement of tyrosine kinases and cyclic nucleotide-dependent protein kinases but to only minor or no contributions of PKC-, PI3K-, and MAPK-dependent signaling in mediating osmosensitivity of Bhmt mRNA expression.

Osmolyte dependence of hyperosmotic Bhmt mRNA suppression. Like hyperosmotic NaCl, increasing osmolarity by the impermeable osmolyte raffinose to 405 mosmol/l reduced Bhmt mRNA levels after 24 h (Fig. 4). However, adjustment of hyperosmolarity with urea, a solute rapidly penetrating liver cells (35), was ineffective to decrease Bhmt mRNA expression levels in H4IIE cells. Similar to hypoosmolarity, urea rather increased Bhmt steady-state mRNA levels (Fig. 3). This may be explained by the earlier report showing that urea in some respects mimics the hepatocellular response to hypoosmolarity (11). Overall, our findings indicate that cell shrinkage rather than increased concentrations of Na+ and Cl– or an upward shift of osmolarity per se triggers suppression of Bhmt mRNA expression levels.


Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 4. Differential effects of hyperosmolarity adjusted by raffinose, urea, or NaCl on Bhmt mRNA expression. H4IIE cells were exposed to hyperosmotic media. Hyperosmolarity of 405 mosmol/l was adjusted by addition of 100 mM raffinose, urea, or 50 mM NaCl. After 24 h, cells were lysed for RNA preparation. Bhmt mRNA expression was determined by quantitative RT-PCR. The figure summarizes data from 3 different experiments. *Relative mRNA expression significantly different from the respective normoosmotic control.

 
Effect of anisoosmolarity on BHMT promoter activity and Bhmt mRNA stability. To study osmosensitivity of BHMT promoter activity, a plasmid containing the entire promoter of the human BHMT gene fused to the luciferase reporter gene was transfected into H4IIE cells. As shown in Fig. 5, hypoosmolarity increased the activity of the human BHMT promoter about fivefold. Hypoosmolarity was less efficient to stimulate transcription driven by a truncated 400-bp fragment of the BHMT promoter (Fig. 5A), indicating additional responsive elements to be localized between the positions –3179 and –400 of the BHMT promoter. Hyperosmolarity tended to decrease BHMT promoter activity under control of the longer fragment while the shorter fragment slightly increased its activity. The findings indicate that hypoosmotic swelling affects BHMT expression at the transcriptional level.


Figure 5
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 5. Anisoosmotic effects on BHMT promoter activity and Bhmt mRNA stability. A: osmosensitive regulation of the human BHMT promoter. H4IIE cells cotransfected with the respective reporter construct and a control construct were exposed to hypoosmotic (Hypo), normoosmotic (Normo), or hyperosmotic (Hyper) medium for 24 h. BHMT promoter activity was addressed as described in MATERIALS AND METHODS. Luciferase activity normalized for transfection efficiency was taken as a measure for BHMT promoter activity and presented relative to that found under the normoosmotic control condition. Shown are means and SEs of 3 independent experiments. *Luciferase activity significantly different from the respective normoosmotic control or between constructs, respectively. The promoter constructs are schematized, for description see MATERIALS AND METHODS. B: effect of anisoomolarity on Bhmt mRNA stability. Bhmt mRNA expression was increased by a 24 h exposure of the H4IIE cells to hypoosmolarity. Then medium was exchanged and cells were incubated for another 30, 60, or 120 min under hypo-, normo-, or hyperosmotic conditions in presence or absence of actinomycin D (act D, 10 µg/ml). Bhmt mRNA expression levels were analyzed by RT-PCR as described in the legend to Fig. 2. Bhmt mRNA levels at the zero time point under the normoosmotic control condition was set to 100%. *Relative Bhmt mRNA expression significantly different from that found under the respective osmotic condition in absence of act D. §Relative Bhmt mRNA expression under the hyperosmolarity plus act D condition differs significantly from that found under hypo- or normoosmotic conditions in the presence of act D.

 
To study a potential effect of anisoosmolarity on Bhmt mRNA stability, experiments with the transcription inhibitor actinomycin D were performed (Fig. 5B). After a hypoosmotic prestimulation of Bhmt mRNA expression, cells were incubated for another 30, 60, or 120 min in presence of actinomycin D under hypo-, normo-, or hyperosmotic conditions, respectively. As shown in Fig. 5B, actinomycin D consistently led to a decay of Bhmt mRNA, irrespective of the ambient osmolarity. At 120 min after actinomycin D addition, Bhmt mRNA levels were significantly decreased under hyperosmotic conditions, compared with the Bhmt mRNA levels found under the respective normo- or hypoosmotic condition. This suggests that Bhmt mRNA destabilization could contribute to the suppression of Bhmt mRNA expression by hyperosmolarity.

Bhmt protein concentrations follow changes in gene expression. Western blot analysis (Fig. 6) showed that the osmosensitivity of Bhmt mRNA expression is also reflected at the protein level. After 24 h, Bhmt expression was increased 1.53 ± 0.21-fold under hypoosmotic and decreased to 0.50 ± 0.13-fold expression under hyperosmotic conditions (n = 4).


Figure 6
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 6. Osmotic regulation of Bhmt protein expression. H4IIE cells were exposed to hypo- (205 mosmol/l), normo- (305 mosmol/l), or hyperosmotic (405 mosmol/l) medium for the time periods indicated. Total cell protein was analyzed for the presence of Bhmt by probing Western blots with an antibody specifically recognizing Bhmt. A representative blot of 4 independent experiments is shown. For densitometric quantification the normalized BHMT expression [ratio of BHMT to glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] under the normoosmotic control condition was taken as onefold. *Bhmt expression significantly (P < 0.05) different from that found under the normoosmotic control condition.

 
Bhmt enzymatic activity and concentrations of its substrate betaine and its product DMG are altered under anisoosmotic conditions. Activity of Bhmt in lysates of H4IIE cells was 5.3 ± 2.3 U/mg protein at ambient osmolarity of 305 mosmol/l. Bhmt activity was 9.1 ± 1.3 U/mg protein after 24 h incubation in 205 mosmol/l and 2.3 ± 1.2 U/mg protein in 405 mosmol/l (n = 3). Both effects were statistically significant (Fig. 7).


Figure 7
View larger version (6K):
[in this window]
[in a new window]

  		Fig. 7. Osmosensitivity of Bhmt activity. H4IIE cells were exposed for 24 h to hypo- (205 mosmol/l), normo- (305 mosmol/l), or hyperosmotic (405 mosmol/l) medium. Bhmt activity was normalized for protein content of lysates. Shown are means and SEs of 3 independent experiments. Expression of mean Bhmt activity in the respective control samples was set to 1. *Relative Bhmt activity significantly different from that found under the normoosmotic control condition.

 
In addition, we measured intra- and extracellular concentrations of betaine and DMG, the substrate and product of the Bhmt-catalyzed reaction, respectively (Fig. 8). Hypoosmolarity (24 h) decreased intracellular and increased extracellular betaine concentrations, indicating a volume-regulatory release of betaine. Simultaneously, both intra- and extracellular DMG concentrations were decreased, suggesting its further metabolization by the DMG dehydrogenase. Hyperosmolarity (24 h) significantly decreased medium betaine concentration. In view of the initial absence of betaine in the medium, the relatively low medium concentration of choline (its metabolic precursor) and the lack of hyperosmotic BGT-1 induction in H4IIE cells (38), this may largely reflect a reduced net release of betaine. Intracellular betaine concentrations were not significantly changed by hyperosmolarity, whereas both intra- and extracellular DMG concentrations were significantly reduced. The intracellular ratio of DMG to betaine was significantly elevated in cells exposed to hypoosmolarity and slightly but not significantly decreased under hyperosmotic conditions, which would correspond to the osmosensitive expression of Bhmt. These data are consistent with the view that betaine acts as an osmolyte in H4IIE cells and that an increase of Bhmt activity contributes to the reduction of betaine levels in H4IIE cells under hypoosmotic conditions, whereas a decrease of Bhmt activity may contribute to save intracellular betaine under hyperosmotic conditions.


Figure 8
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 8. Intracellular concentrations of betaine and dimethylglycine (DMG) in H4IIE cells exposed to anisoosmolarity. H4IIE cells were exposed for 24 h to hypo- (205 mosmol/l), normo- (305 mosmol/l), or hyperosmotic (405 mosmol/l) culture medium, which was virtually free of betaine. Betaine and DMG concentrations in cell lysates and the medium after 24 h were determined by HPLC-tandem mass spectrometry as described in MATERIALS AND METHODS. Betaine and DMG in lysates were normalized for protein content. Shown are means and SEs of 5 independent experiments. *Significant deviation from normoosmotic control conditions.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study demonstrates that Bhmt expression in H4IIE rat hepatoma cells is strongly regulated by changes in ambient osmolarity. Bhmt mRNA and protein expression and Bhmt enzyme activity are upregulated under hypoosmotic but downregulated under hyperosmotic conditions. Increased net release and catabolic elimination of betaine decrease the intracellular concentration of betaine and may facilitate the adaptation to a hypoosmotic extracellular ambience. Hyperosmolarity induces the opposite response.

Bhmt expression and activity were decreased also in vivo in the liver and kidney of NaCl-loaded guinea pigs (7). Interestingly, NaCl treatment was without effect on pancreatic Bhmt activity (7). Serum osmolarity was not monitored in this study, and indirect mechanisms triggered by, e.g., altered cytokine or hormone patterns on Bhmt expression cannot be excluded in this model. The use of cultured cells exposed to anisoosmotic media in the study presented here avoids such confounding variables possibly caused by secondary effects of high-salt animal treatment. As shown here (Fig. 4), cell shrinkage as induced by addition of NaCl or raffinose was sufficient to downregulate BHMT expression, whereas urea was ineffective. Thus neither elevation of ionic strength nor an osmolarity increase per se accounts for downregulation of Bhmt. Osmotic regulation of Bhmt mRNA expression was found in both, hepatoma cells and cultured hepatocytes (Fig. 2), suggesting that it is not related to cell transformation.

The pharmacological characterization performed in this study (Fig. 3) supports the involvement of genistein-sensitive tyrosine kinases and cyclic nucleotide-dependent protein kinases in mediating the osmosensitivity of Bhmt mRNA expression. Interestingly, cyclic nucleotide-dependent protein kinases but not MAPKs, PKC, PI3K and mTOR were suggested in H4IIE cells to also play role in mediating the osmosensitivity of phosphoenolpyruvate carboxykinase mRNA expression, which, in contrast to Bhmt mRNA (this study), is suppressed under hypoosmotic conditions and induced under hyperosmotic conditions (22, 37).

The data presented in Fig. 5 support the view that hypoosmolarity increases Bhmt mRNA levels by stimulation of transcription, whereas destabilization of Bhmt mRNA may play a major part in its decrease under hyperosmotic conditions. To facilitate the examination of the Bhmt mRNA decay, Bhmt mRNA levels were increased by a hypoosmotic treatment before the addition of actinomycin D. Therefore, at this stage, it cannot be excluded that the hypoosmotic pretreatment per se modifies the osmosensitivity of Bhmt mRNA stability. Furthermore, indirect effects of actinomycin D [e.g., by preventing expression of Bhmt mRNA (de)stabilizing proteins] could be taken into consideration.

What functional role could the osmotic regulation of Bhmt activity play in the liver? In the liver, parenchymal cells synthesize betaine, which is utilized by nonparenchymal endothelial cells (39), Kupffer cells (44), and stellate cells (25) as an osmolyte. Thus inactivation of parenchymal Bhmt under dehydrating conditions may make higher amounts of betaine available for protection of the nonparenchymal cells. On the other hand, a hypoosmotic stimulation of betaine catabolism may contribute to a regulatory volume decrease. Besides its role in volume regulation, betaine has an important metabolic role in hepatic homocysteine metabolism. Betaine catabolism indirectly provides methyl groups for many essential transmethylation reactions in the body. Cell swelling is associated with increased anabolism, whereas cellular dehydration confers a catabolic situation (12). Increased Bhmt activity under conditions of cell swelling may lead to an increased provision of betaine-derived methyl groups for synthetic pathways. On the other hand, hyperosmotic dehydration sensitizes hepatocytes and H4IIE cells to different kinds of stress including apoptotic stimuli (19, 27), and this may be supported by a deficiency of betaine-derived methyl groups due to BHMT inactivation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Deutsche Forschungsgemeinschaft through Sonderforschungsbereich 575 "Experimentelle Hepatologie" (Düsseldorf) and by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50521.


   ACKNOWLEDGMENTS
 
The technical assistance of Nicole Rustige, Andrew P. Breska III, and Jana Srakova is gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. Schliess, Universitätsklinikum Düsseldorf, Klinik für Gastroenterologie, Hepatologie und Infektiologie, Moorenstrasse 5, D-40225 Düsseldorf, Germany (e-mail: schliess{at}med.uni-duesseldorf.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.

* C. Schäfer and L. Hoffmann contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 262: 5592–5595, 1987.[Abstract/Free Full Text]
  2. Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR. PD098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 270: 27489–27494, 1995.[Abstract/Free Full Text]
  3. Bennett BL, Sasaki DT, Murray BW, O'Leary EC, Sakata ST, Xu W, Leisten JC, Motiwala A, Pierce S, Satoh Y, Bhagwat SS, Manning AM, Anderson DW. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci USA 98: 13681–13686, 2001.[Abstract/Free Full Text]
  4. Burg MB, Kwon ED, Kultz D. Regulation of gene expression by hypertonicity. Annu Rev Physiol 59: 437–455, 1997.[CrossRef][ISI][Medline]
  5. Cheatham B, Vlahos CJ, Cheatham L, Wang L, Blenis J, Kahn CR. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of p70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol 14: 4902–4911, 1994.[Abstract/Free Full Text]
  6. Cross DA, Alessi DR, Vandenheede JR, McDowell HE, Hundal HS, Cohen P. The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf. Biochem J 303: 21–26, 1994.[ISI][Medline]
  7. Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanisms of action of some commonly used protein kinase inhibitors. Biochem J 351: 95–105, 2000.[CrossRef][ISI][Medline]
  8. Delgado-Reyes CV, Garrow TA. High sodium chloride intake decreases betaine-homocysteine S-methyltransferase expression in guinea pig liver and kidney. Am J Physiol Regul Integr Comp Physiol 288: R182–R187, 2005.[Abstract/Free Full Text]
  9. Findik D, Song Q, Hidaka H, Lavin M. Protein kinase A inhibitors enhance radiation-induced apoptosis. J Cell Biochem 57: 12–21, 1995.[CrossRef][ISI][Medline]
  10. Forestier M, Banninger R, Reichen J, Solioz M. Betaine homocysteine methyltransferase: gene cloning and expression analysis in rat liver cirrhosis. Biochim Biophys Acta 1638: 29–34, 2003.[Medline]
  11. Garrow TA. Purification, kinetic properties, and cDNA cloning of mammalian betaine-homocysteine methyltransferase. J Biol Chem 271: 22831–22838, 1996.[Abstract/Free Full Text]
  12. Hallbrucker C, vom Dahl S, Ritter M, Lang F, Häussinger D. Effects of urea on K+ fluxes and cell volume in perfused rat liver. Pflügers Arch 428: 552–560, 1994.[CrossRef][ISI][Medline]
  13. Häussinger D, Kubitz R, Reinehr R, Bode JG, Schliess F. Molecular aspects of medicine: from experimental to clinical hepatology. Mol Aspects Med 25: 221–360, 2004.[CrossRef][Medline]
  14. Ho AK, Chik CL, Klein DC. Effects of protein kinase inhibitor (1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) on protein kinase C activity and adrenergic stimulation of cAMP and cGMP in rat pinealocytes. Biochem Pharmacol 37: 1015–1020, 1988.[CrossRef][ISI][Medline]
  15. Jackson JR, Bolognese B, Hillegass L, Kassis S, Adams J, Griswold DE, Winkler JD. Pharmacological effects of SB 220025, a selective inhibitor of P38 mitogen-activated protein kinase, in angiogenesis and chronic inflammatory disease models. J Pharmacol Exp Ther 284: 687–692, 1998.[Abstract/Free Full Text]
  16. Lang F, Busch GL, Ritter M, Völkl H, Waldegger S, Gulbins E, Häussinger D. Functional significance of cell volume regulatory mechanisms. Physiol Rev 78: 247–306, 1998.[Abstract/Free Full Text]
  17. Laryea MD, Steinhagen F, Pawliczek S, Wendel U. Simple method for the routine determination of betaine and N,N-dimethylglycine in blood and urine. Clin Chem 44: 1937–1941, 1998.[Abstract/Free Full Text]
  18. Liu Y, Bolen DW. The peptide backbone plays a dominant role in protein stabilization by naturally occurring osmolytes. Biochemistry 34: 12884–12891, 1995.[CrossRef][Medline]
  19. Lornejad-Schäfer MR, Schäfer C, Graf D, Häussinger D, Schliess F. Osmotic regulation of insulin-induced mitogen-activated protein kinase phosphatase (MKP-1) expression in H4IIE rat hepatoma cells. Biochem J 371: 609–619, 2003.[CrossRef][ISI][Medline]
  20. Lornejad-Schäfer MR, Schäfer C, Richter L, Grune T, Häussinger D, Schliess F. Osmotic regulation of MG-132-induced MAP-kinase phosphatase MKP-1 expression in H4IIE rat hepatoma cells. Cell Physiol Biochem 16: 193–206, 2005.[CrossRef][ISI][Medline]
  21. Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, Hug H, Marmé D, Schächtele G. Selective inhibition of protein kinase C isoenzymes by the indolocarbazole Gö6976. J Biol Chem 268: 9194–9197, 1993.[Abstract/Free Full Text]
  22. Millian NS, Garrow TA. Human betaine-homocysteine methyltransferase is a zinc metalloenzyme. Arch Biochem Biophys 356: 93–98, 1998.[CrossRef][ISI][Medline]
  23. Newsome WP, Warskulat U, Noé B, Wettstein M, Stoll B, Gerok W, Häussinger D. Modulation of phosphoenolpyruvate carboxykinase levels by the hepatocellular hydration state. Biochem J 304: 555–560, 1994.[ISI][Medline]
  24. Park EI, Garrow TA. Interaction between dietary methionine and methyl donor intake on rat liver betaine-homocysteine methyltransferase gene expression and organization of the human gene. J Biol Chem 274: 7816–7824, 1999.[Abstract/Free Full Text]
  25. Park EI, Renduchintala MS, Garrow TA. Diet-induced changes in hepatic betaine homocysteine methyltransferase activity are mediated by changes in the steady-state level of its mRNA. J Nutr Biochem 8: 541–545, 1997.[CrossRef][ISI]
  26. Peters-Regehr T, Bode JG, Kubitz R, Häussinger D. Organic osmolyte transport in quiescent and activated rat hepatic stellate cells (Ito cells). Hepatology 29: 173–180, 1999.[CrossRef][ISI][Medline]
  27. Reinehr R, Graf D, Häussinger D. Bile salt-induced hepatocyte apoptosis involves epidermal growth factor receptor-dependent CD95 tyrosine phosphorylation. Gastroenterology 125: 839–853, 2003.[CrossRef][ISI][Medline]
  28. Reinehr RM, Graf D, Fischer R, Schliess F, Häussinger D. Hyperosmolarity triggers CD95 membrane trafficking and sensitizes rat hepatocytes towards CD95L-induced apoptosis. Hepatology 36: 602–614, 2002.[CrossRef][ISI][Medline]
  29. Schliess F, Häussinger D. The cellular hydration state: role in apoptosis and proliferation. Signal Transduction 6: 297–302, 2005.
  30. Schliess F, Heinrich S, Häussinger D. Hyperosmotic induction of the mitogen-activated protein kinase phosphatase MKP-1 in H4IIE rat hepatoma cells. Arch Biochem Biophys 351: 35–40, 1998.[CrossRef][ISI][Medline]
  31. Schwahn BC, Chen Z, Laryea MD, Wendel U, Lussier-Cacan S, Genest JJ, Mar MH, Zeisel SH, Castro C, Garrow T, Rozen R. Homocysteine-betaine interactions in a murine model of 5,10-methylenetetrahydrofolate reductase deficiency. FASEB J 17: 512–514, 2003.[Abstract/Free Full Text]
  32. Schwahn BC, Laryea MD, Chen Z, Melnyk S, Pogribny I, Garrow T, James SJ, Rozen R. Betaine rescue of an animal model with methylenetetrahydrofolate reductase deficiency. Biochem J 382: 831–840, 2004.[CrossRef][ISI][Medline]
  33. Schwahn BC, Wendel U, Lussier-Cacan S, Mar MH, Zeisel SH, Leclerc D, Castro C, Garrow TA, Rozen R. Effects of betaine in a murine model of mild cystathionine-beta-synthase deficiency. Metabolism 53: 594–599, 2004.[CrossRef][ISI][Medline]
  34. Sheikh-Hamad D, Garcia-Perez A, Ferraris JD, Peters EM, Burg MB. Induction of gene expression by heat shock vs. osmotic stress. Am J Physiol Renal Fluid Electrolyte Physiol 267: F28–F34, 1994.[Abstract/Free Full Text]
  35. Sunden SL, Renduchintala MS, Park EI, Miklasz SD, Garrow TA. Betaine-homocysteine methyltransferase expression in porcine and human tissues and chromosomal localization of the human gene. Arch Biochem Biophys 345: 171–174, 1997.[CrossRef][ISI][Medline]
  36. Vom Dahl S, Hallbrucker C, Lang F, Gerok W, Häussinger D. A non-invasive technique for cell volume determination in perfused rat liver. Biol Chem Hoppe Seyler 372: 411–418, 1991.[ISI][Medline]
  37. Warskulat U, Newsome W, Noé B, Stoll B, Häussinger D. Anisoosmotic regulation of hepatic gene expression. Biol Chem Hoppe Seyler 377: 57–65, 1996.[ISI][Medline]
  38. Warskulat U, Wettstein M, Häussinger D. Osmoregulated taurine transport in H4IIE cells and perfused rat liver. Biochem J 321: 683–690, 1997.[ISI][Medline]
  39. Weik C, Warskulat U, Bode JG, Peters-Regehr T, Häussinger D. Compatible organic osmolytes in rat liver sinusoidal endothelial cells. Hepatology 27: 569–575, 1998.[CrossRef][ISI][Medline]
  40. Wendel U, Bremer HJ. Betaine in the treatment of homocystinuria due to 5,10-methylenetetrahydrofolate reductase deficiency. Eur J Pediatr 142: 147–150, 1984.[CrossRef][ISI][Medline]
  41. Wettstein M, Häussinger D. Cytoprotection by the osmolytes betaine and taurine in ischemia-reoxygenation injury in the perfused rat liver. Hepatology 26: 1560–1566, 1997.[ISI][Medline]
  42. Wiese S, Schliess F, Häussinger D. Osmotic regulation of MAP-kinase activities and gene expression in H4IIE rat hepatoma cells. Biol Chem 379: 667–671, 1998.[ISI][Medline]
  43. Wilken DR, McMacken ML, Rodriquez A. Choline and betaine aldehyde oxidation by rat liver mitochondria. Biochim Biophys Acta 216: 305–317, 1970.[Medline]
  44. Zhang F, Warskulat U, Wettstein M, Häussinger D. Identification of betaine as an osmolyte in rat liver macrophages (Kupffer cells). Gastroenterology 110: 1543–1552, 1996.[CrossRef][ISI][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/4/G1089    most recent
00088.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schäfer, C.
Right arrow Articles by Schliess, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schäfer, C.
Right arrow Articles by Schliess, F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.