Cécile Mazière
email: maziere.cecile@chu-amiens.fr

Valéry Savitsky

Antoine Galmiche

Cathy Gomila

Ziad Massy

Jean-Claude Mazière

Oxidized low density lipoprotein inhibits phosphate signaling and phosphateinduced mineralization in osteoblasts. Involvement of oxidative stress

Keywords: osteoblasts, differentiation, OxLDL, oxidative stress, signaling Inorganic phosphate, OxLDL: Cu 2+ -oxidized low density lipoprotein, OPN: osteopontin, RANKL: Receptor Activator of NFkappaB ligand, ERK: Extracellular signal-Regulated Kinase, JNK: Jun kinase, AP1: Activator Protein 1

BACKGROUND: It is well admitted that oxidized LDL (OxLDL) plays a major role in the generation and progression of atherosclerosis. Since atherosclerosis is often accompanied by osteoporosis, the effects of OxLDL on phosphate-induced osteoblast mineralization were investigated. METHODS: Calcium deposition, expression of osteoblast markers and inorganic phosphate (Pi) signaling were determined under OxLDL treatment. RESULTS: OxLDL, within the range of 10-50 µg protein/ml, inhibited Pi-induced UMR106 rat osteoblast mineralization. In parallel, the expression of Cbfa1/Runx2 transcription factor was decreased, and the intracellular level of the osteoblast marker osteopontin (OPN) was reduced. The extracellular level of another marker, Receptor Activator of Nuclear Factor kappa B Ligand (RANKL), was also diminished. OxLDL inhibited Pi signaling via ERK/JNK kinases and AP1/CREB transcription factors. OxLDL triggered the generation of reactive oxygen species (ROS), either in the absence or presence of Pi. Furthermore, the effects of OxLDL on Piinduced mineralization, generation of ROS and extracellular level OPN were reproduced by the lipid extract of the particle, whereas the antioxidant vitamin E prevented them. CONCLUSIONS: This work demonstrates that OxLDL, by generation of an oxidative stress, inhibits of Pi signaling and impairs Pi-induced osteoblast differentiation. GENERAL SIGNIFICANCE: This highlights the role of OxLDL in bone remodeling and in degenerative disorders other than atherosclerosis, especially in osteoporosis.

Introduction

Bone mass results from an active remodeling process which involves an equilibrium between mineralization, mediated by osteoblasts, and resorption, mediated by osteoclasts.

During osteoblast differentiation, the expression of osteoblast marker genes such as alkaline phosphatase and osteocalcin [START_REF] Beck | Relationship between alkaline phosphatase levels, osteopontin expression, and mineralization in differentiating MC3T3-E1 osteoblasts[END_REF], or osteopontin OPN [START_REF] Hsu | Diosmetin induces human osteoblastic differentiation through the protein kinase C/p38 and extracellular signal-regulated kinase 1/2 pathway[END_REF][START_REF] Zirngibl | Estrogen receptor-related receptor alpha (ERRalpha) regulates osteopontin expression through a non-canonical ERRalpha response element in a cell context-dependent manner[END_REF] are enhanced. A crosstalk between osteoblasts and osteoclasts exists in that osteoblast differentiation is accompanied by expression of RANKL (Receptor Activator of Nuclear Factor kappa B Ligand) (4), a cytokine involved in osteoclast differentiation. Conversely, inorganic phosphate (Pi) released by bone resorption is a signaling molecule in the events preceeding mineralization of osteoblasts [START_REF] Beck | Inorganic phosphate regulates multiple genes during osteoblast differentiation, including Nrf2[END_REF]. In MC3T3-E1 mouse osteoblasts, it has been demonstrated that Pi induced OPN gene expression via ERK and PKC kinases [START_REF] Beck | Osteopontin regulation by inorganic phosphate is ERK1/2, protein kinase C-, and proteasome-dependent[END_REF]. Other kinases have also been demonstrated to mediate osteoblast differentiation. In this regard, PKD activation mediates Bone Morphogenetic Protein 2 BMP2 and Insulin-like Growth Factor 1 IGF1 treatment [START_REF] Celil | BMP-2 and insulin-like growth factor-I mediate Osterix (Osx) expression in human mesenchymal stem cells via the MAPK and protein kinase D signaling pathways[END_REF], and p38 mediates diosmetin treatment [START_REF] Matsuguchi | JNK Activity is essential for Atf4 Expression and Late-Stage Osteoblast Differentiation[END_REF].

JNK is also involved in late-stage osteoblast differentiation [START_REF] Hsu | Diosmetin induces human osteoblastic differentiation through the protein kinase C/p38 and extracellular signal-regulated kinase 1/2 pathway[END_REF].

Concerning the transcription factors, Runx2/Cbfa1/Osf2 plays a crucial role in bone mineralization [START_REF] Ducy | Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation[END_REF]. The activity of this factor is regulated by both gene expression and posttranslational regulation. The expression of Runx2 was demonstrated to be under the control of PKC [START_REF] Chen | High glucose increases the expression of Cbfa1 and BMP-2 and enhances the calcification of vascular smooth muscle cells[END_REF] and p38 [START_REF] Lee | Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein[END_REF]. In addition, the binding activity of Runx2 is enhanced by phosphorylation via Ras/Raf/ERK1/2 pathway [START_REF] Kanno | Mechanical stressmediated Runx2 activation is dependent on Ras/ERK1/2 MAPK signaling in osteoblasts[END_REF]. Besides Runx2, AP1 activation has also been described in BMP2-induced bone formation [START_REF] Lai | Signal transductions induced by bone morphogenetic protein-2 and transforming growth factor-beta in normal human osteoblastic cells[END_REF][START_REF] Wu | Naringin-induced bone morphogenetic protein-2 expression via PI3K, Akt, c-Fos/c-Jun and AP-1 pathway in osteoblasts[END_REF]. In addition, CREB (cyclic AMP Response Element Binding Protein) phosphorylation and transactivation has been demonstrated in TNF -induced mineralization [START_REF] Tintut | Tumor necrosis factor-alpha promotes in vitro calcification of vascular cells via the cAMP pathway[END_REF]. Direct interactions between Runx2 and other transcription factors such as AP1 and Smads, which results in binding to separate sites of DNA, were also described [START_REF] Franceschi | Multiple signaling pathways converge on the Cbfa1/Runx2 transcription factor to regulate osteoblast differentiation[END_REF].

It is well established that oxidized LDL (OxLDL) plays a major role in the generation and progression of the atherosclerotic plaque (Rev in 17). It has been further observed that atherosclerosis is often accompanied by osteoporosis [START_REF] Hofbauer | Vascular calcification and osteoporosis--from clinical observation towards molecular understanding[END_REF]. We recently reported a role for OxLDL in bone remodeling in that these particles impair RANKL signaling and RANKLinduced osteoclast differentiation [START_REF] Mazière | Oxidized low density lipoprotein decreases Rankl-induced differentiation of osteoclasts by inhibition of Rankl signalling[END_REF]. In addition, it was reported that minimally oxidized LDL and lipid oxidation products, such as oxidized phosphatidyl-choline and isoprostane8-iso prostaglandin E2, inhibit bone cell differentiation [START_REF] Parhami | Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. A possible explanation for the paradox of arterial calcification in osteoporotic patients[END_REF]. This effect of OxLDL has been

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attributed to generation of an oxidative stress [START_REF] Mody | Oxidative stress modulates osteoblastic differentiation of vascular and bone cells[END_REF]. However, the molecular mechanism of this inhibitory effect was not yet described. We previously demonstrated that OxLDL inhibits insulin signaling and that this effect might be ascribed to the generation of an oxidative stress [START_REF] Mazière | Inhibition of insulin signaling by oxidized low density lipoprotein. Protective effect of the antioxidant Vitamin E[END_REF].

Several reports mentioned the sensitivity of osteoblasts towards oxidative stress [START_REF] Fatokun | Responses of differentiated MC3T3-E1 osteoblastlike cells to reactive oxygen species[END_REF][START_REF] Lee | Myricetin, a naturally occurring flavonoid, prevents 2-deoxy-Dribose induced dysfunction and oxidative damage in osteoblastic MC3T3-E1 cells[END_REF], and especially, osteoblasts signaling pathways such as Wnt pathway might be impaired [START_REF] Almeida | Oxidative stress antagonizes Wnt signaling in osteoblast precursors by diverting beta-catenin from T cell factorto forkhead box O-mediated transcription[END_REF]. In the current study, we demonstrate that OxLDL prevented Pi-induced differentiation of osteoblasts and inhibited Pi signaling through ERK/JNK kinases, and AP1/CREB transcription factors.

Furthermore, this phenomenon might be ascribed to the generation of reactive oxygen species ROS, since the antioxidant vitamin E prevented it.

Materials and methods

Cell culture

UMR106 rat osteoblasts were purchased from the American Type Culture Collection. Elisa kits for rat OPN was from IBL, Hambourg, Deutchland, and for rat RANKL from Biomedica, Vienne, Austria. Determination of intracellular OPN was performed on 2 µg protein.

Secreted OPN was determined after 1/100 dilution of culture medium and secreted RANKL was measured after concentration by a factor 5 using Microcon tubes from Millipore.

For the study of Pi signaling, cells were preincubated 24h in medium devoid of serum and supplemented with 0.1% bovine albumin in order to down regulate all signaling pathways.

OxLDL were then introduced for 4h and 4 mM Pi was added for 45 min. Cellular or nuclear extracts were then performed for determination of kinase phosphorylation by western blot, and transcription factor DNA binding activities by Elisa.

Visualization and determination of calcium deposition.

Cells were treated in 48 well-plates and calcification was visualized using the Von Kossa staining technique in the presence of AgNO 3 for phosphate deposition [START_REF] Towler | Diet-induced diabetes activates an osteogenic gene regulatory program in the aortas of low density lipoprotein receptor-deficient mice[END_REF]. Cells were fixed

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Determination of intracellular OPN, Cbfa1/Runx2 expression and ERK/JNK

phosphorylation by immunoblot analysis.

Cells were lysed in 50 mM Tris, 50 mM NaF, 20 mM p-nitrophenyl phosphate, 1 mM EGTA, 0.05 mM Na-vanadate, 5 mM benzamidine and 1% TX100 during 15 min at 4°C and sonicated. After centrifugation at 14 000 g for 2 min, the supernatants were taken as cell lysates.

Equal amounts of proteins (40-50 µg) were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with specific antibodies. Equal loading was checked by neutral red staining. Immunoblots were visualized by enhanced chemi-luminescence detection kit from Amersham (Saclay, France). The OPN antibody was from Santa Cruz (CA, USA), Cbfa1/Runx2 antibody from Calbiochem (CA,USA) and ERK/JNK antibodies were from Cell Signaling (MA, USA).

Determination of AP1/CREB DNA binding activities by ELISA

The binding activity assays were performed using the Trans-AM ELISA-based kits from Active Motif (Carlsbad, CA) according to the manufacturer's protocol. Nuclear extracts were prepared using Active Motif kits. Equal amounts of proteins were incubated in 96-well plates coated with oligonucleotides containing the AP1 or CREB consensus binding sites. Activated The increase in fluorescence in living cells in the presence of this probe allows the quantitation of ROS such as superoxide anion, hydrogen peroxide and the hydroxyl radical [START_REF] Scott | Quantitation of intracellular oxidation in a renal epithelial cell line[END_REF].

Cells in 3.5 cm Petri dishes were incubated for 15 min with 10 -5 M chloro-methyl-2'7'dichlorofluorescein (Molecular Probes) in PBS, washed three times with PBS, solubilized in H 2 O and sonicated. The fluorescence was determined at 503/529 nm, normalized on a protein basis and expressed as % of control.

Results

OxLDL inhibited Pi-induced osteoblast mineralization.

We first studied the effect of OxLDL on mineralization of UMR106 rat osteoblasts, 12

OxLDL reduced Pi-induced expression of OPN and RANKL.

We then investigated the effects of OxLDL on the expression of specific osteoblast indicate that a 4h incubation with 4 mM Pi initiated a slight but significant increase (x 1.4) in ROS. Furthermore, OxLDL 10-50 µg/ml increased the generation of ROS in a dose-dependent manner, both in the absence or presence of Pi.

The lipid extract of OxLDL reproduced whereas vitamin E prevented the effects of

OxLDL.

We then investigated the effects of the lipid peroxidation products included in OxLDL.

The data from Fig 6 indicate that the lipid extract from OxLDL, like the particle itself, increased

Pi-induced generation of ROS (6A) and simultaneously reduced mineralization (6B) and OPN level (6C). By contrast, the antioxidant vitamin E partially but significantly reversed these effects of OxLDL and its lipid extract.

Discussion

In this study, we first demonstrated that OxLDL inhibited Pi-induced mineralization of . This is in accordance with the report from Parhami et al [START_REF] Parhami | Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. A possible explanation for the paradox of arterial calcification in osteoporotic patients[END_REF], which described an inhibitory effect of minimally-oxidized LDL and of oxidized phosphatidyl-choline on bone cell differentiation. These authors utilized minimally-iron-oxidized LDL at the concentration of 200 µg LDL protein/ml, which is equivalent, in terms of lipid peroxidation products (TBARS), to the highest concentration used in our experimental model (50 µg LDL protein/ml). They further demonstrated that minimally-oxidized LDL inhibits osteogenic differentiation of marrow stromal cells by directing these cells to undergo adipogenic differentiation by activation of PPAR alpha [START_REF] Parhami | Atherogenic diet and minimally oxidized low density lipoprotein inhibit osteogenic and promote adipogenic differentiation of marrow stromal cells[END_REF]. However, these authors only determined alkaline phosphatase as osteoblast marker, and furthermore, the effect of OxLDL on osteoblast signaling pathways was not yet reported.

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The current studies demonstrate that in rat osteoblasts, Pi signaling involves ERK/JNK kinases (Fig3), and AP1/CREB transcription factors (Fig 4). Pi transduction signal via ERK in mouse osteoblasts [START_REF] Beck | Osteopontin regulation by inorganic phosphate is ERK1/2, protein kinase C-, and proteasome-dependent[END_REF] or via AP1 in BALB/c mouse JB6 epidermal cells (33) was previously reported. Furthermore, OxLDL alone exhibited the same stimulatory effect, in accordance with previous reports concerning ERK [START_REF] Augé | Potential role for ceramide in mitogen-activated protein kinase activation and proliferation of vascular smooth muscle cells induced by oxidized low density lipoprotein[END_REF], JNK [START_REF] Schroeter | Flavonoids protect neurons from oxidized low-density-lipoprotein-induced apoptosis involving c-Jun N-terminal kinase (JNK), c-Jun and caspase-3[END_REF] and AP1 [START_REF] Mazière | Copper and cell-oxidized low-density lipoprotein induces activator protein 1 in fibroblasts, endothelial and smooth muscle cells[END_REF]. However, preincubation with OxLDL prevented Pi signaling, thus demonstrating a crosstalk between both activation mechanisms.

It is well admitted that ERK controls AP1 [START_REF] Benkoussa | Retinoic acid receptors inhibit AP1 activation by regulating extracellular signal-regulated kinase and CBP recruitment to an AP1-responsive promoter[END_REF] and Cbfa1/Runx2 (38) activities. In addition, the expression of OPN gene was also demonstrated to be under the control of ERK in smooth muscle cells [START_REF] Moses | Injury-induced osteopontin gene expression in rat arterial smooth muscle cells is dependent on mitogen-activated protein kinases ERK1/ERK2[END_REF] or cardiac fibroblasts [START_REF] Xie | ERK1/2 and JNKs, but not p38 kinase, are involved in reactive oxygen species-mediated induction of osteopontin gene expression by angiotensin II and interleukin-1beta in adult rat cardiac fibroblasts[END_REF]. It is also of note that ERK mediates OPN expression in arterial smooth muscle cells by activation of AP1 [START_REF] Bidder | Osteopontin transcription in aortic vascular smooth muscle cells is controlled by glucose-regulated upstream stimulatory factor and activator protein-1 activities[END_REF] and CREB [START_REF] Jalvy | Autocrine expression of osteopontin contributes to PDGF-mediated arterial smooth muscle cell migration[END_REF].

Concerning the role of JNK in osteoblast differentiation, it was recently reported that it is essential in the late stage of this phenomenon [START_REF] Matsuguchi | JNK Activity is essential for Atf4 Expression and Late-Stage Osteoblast Differentiation[END_REF], and mediates OPN gene expression [START_REF] Jalvy | Autocrine expression of osteopontin contributes to PDGF-mediated arterial smooth muscle cell migration[END_REF]. Finally, it has been demonstrated that CREB is one of the transcription factors involved in RANKL expression [START_REF] Mak | Hedgehog signaling in mature osteoblasts regulates bone formation and resorption by controlling PTHrP and RANKL expression[END_REF]. Thus, it is reasonable to assume that the inhibitory effect of OxLDL on OPN and RANKL gene expression is related to an impairment of Pi signaling pathways.

Brodeur et al [START_REF] Brodeur | Influence of oxidized lowdensity lipoproteins (LDL) on the viability of osteoblastic cells[END_REF] reported that low concentrations of OxLDL (10-50 µg/ml) induced proliferation of osteoblasts whereas high concentrations (above 150 µg/ml) were cytotoxic. In our experimental model, generation of ROS and impairment of Pi signaling were observed after short-term (4h) incubation with OxLDL, and thus can not be ascribed to a cytotoxic effect of these particles. Furthermore, even after 4 days incubation with Pi and 10-50 µg/ml OxLDL, the experimental conditions chosen for the study of OPN and RANKL expression, no cytotoxicity could be detected in our model, as assessed by the Neutral Red uptake method (data not shown).

It was further demonstrated that Pi induced a significant increase in ROS (Fig 5), which is in accordance with the report from Shuto et al. on aortic endothelial cells [START_REF] Shuto | Dietary phosphorus acutely impairs endothelial function[END_REF]. Furthermore, OxLDL or its lipid extract triggered this effect, whereas the antioxidant vitamin E prevented it (Fig 6). In this regard, it can be noted that oxidized phosphatidyl-choline, a lipid component of OxLDL, has been reported to inhibit spontaneous osteogenic differentiation of marrow stromal cells [START_REF] Parhami | Atherogenic diet and minimally oxidized low density lipoprotein inhibit osteogenic and promote adipogenic differentiation of marrow stromal cells[END_REF] and to decrease osteogenic signaling induced by Bone Morphogenetic Protein 2 and parathyroid hormone in osteoblasts [START_REF] Huang | Atherogenic phospholipids attenuate osteogenic signaling by BMP-2 and parathyroid hormone in osteoblasts[END_REF]. Concerning the mechanism whereby OxLDL initiate generation of ROS, a recent report pointed at the role of CD36 scavenger receptor and NADPH oxidase [START_REF] Park | CD36 modulates migration of mouse and human macrophages in response to oxidized LDL and may contribute to macrophage trapping in the arterial intima[END_REF] in macrophages, whereas the contribution of lipoxygenase and of the mitochondrial

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pathways has been described in vascular smooth muscle cells [START_REF] Hsieh | Oxidized low density lipoprotein induces apoptosis via generation of reactive oxygen species in vascular smooth muscle cells[END_REF]. Finally in osteoblasts, Hamel et al [START_REF] Hamel | Characterization of oxidized low-density lipoprotein-induced hormesis-like effects in osteoblastic cells[END_REF] reported the role of NADPH oxidase in OxLDL-induced ROS generation.

Our studies demonstrate that a moderate generation of ROS, initiated by a single agent, in our case by OxLDL or Pi, activates signaling kinases and transcription factors. By contrast, a too high level of ROS, initiated by OxLDL and 4h later by Pi, might induce a negative effect by generation of an "overoxidized" state. In this regard, it is of note that the oxidative stress caused by hydrogen peroxide also inhibits Pi-induced mineralization in our experimental system (data not shown).

It is well known that OxLDL might inhibit hormone and growth factor signaling pathways, most probably by generation of ROS. In this regard, it was demonstrated that these particles prevent insulin signaling in fibroblasts [START_REF] Mazière | Inhibition of insulin signaling by oxidized low density lipoprotein. Protective effect of the antioxidant Vitamin E[END_REF], PDGF transduction in vascular cells [START_REF] Vindis | Desensitization of platelet-derived growth factor receptor-beta by oxidized lipids in vascular cells and atherosclerotic lesions: prevention by aldehyde scavengers[END_REF] or RANKL transduction signal in osteoclasts [START_REF] Mazière | Oxidized low density lipoprotein decreases Rankl-induced differentiation of osteoclasts by inhibition of Rankl signalling[END_REF]. It can thus be supposed that OxLDL might impair not only Pi signaling but also other transduction pathways crucial for osteoblast differentiation. It must be also noted that osteoblasts have the capacity to oxidize LDL [START_REF] Brodeur | Influence of oxidized lowdensity lipoproteins (LDL) on the viability of osteoblastic cells[END_REF], and that OxLDL are thus generated inside bone matrix.

In conclusion, our studies demonstrate that OxLDL inhibits Pi signaling and Pi-induced mineralization of osteoblasts via generation of an oxidative stress. OxLDL finally down regulates the expression of osteoblast specific genes such as Cbfa1/Runx2, OPN and RANKL.

This highlights the negative role of OxLDL in bone formation, and points at its harmful effect in degenerative pathologies such as osteoporosis. Results are from a typical experiment. This experiment was performed 3 times with similar results.

  Cells were cultured in DMEM containing 10% foetal calf serum. For induction of mineralization, cells were shifted to medium supplemented with 5% foetal calf serum and 4 mM Pi (NaH 2 PO4 in 0.2M stock solution equilibrated to pH 7.4), taking into account the phosphate content of the medium (1 mM). OxLDL 10-50 µg protein/ml, and in some experiments 50 µg/ml OxLDL lipid extract and 50 µM of the antioxidant vitamin E ( -tocopherol from Sigma) were introduced simultaneously with Pi for 4 days.

2 . 6

 26 A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 6 transcription factors bound to the respective immobilized oligonucleotides were detected using the antibodies directed against c-jun or CREB, and a secondary antibody conjugated to horseradish peroxidase. The developing solution was then added for 10 min at room temperature and OD were determined by a microplate reader from Molecular Devices. Results are expressed in % of the OD obtained with non treated cells. Determination of Reactive Oxygen Species ROS with Chloro-Methyl-Dichlorofluorescein.

days after 4 mM

 4 Pi addition. It was observed that OxLDL, within the range of 10-50 µg protein/ml, reduced calcification in a dose dependent manner (Fig 1A), and decreased Piinduced Cbfa1/Runx2 intracellular level (Fig 1B). OxLDL by itself, in the absence of Pi, exhibited no effect. Furthermore, native LDL did not inhibit Pi-induced mineralization (result not shown).

. 3 . 3 3 . 4

 3334 markers. The data from Fig2Ashow that Pi, as previously described in mouse osteoblasts[START_REF] Beck | Osteopontin regulation by inorganic phosphate is ERK1/2, protein kinase C-, and proteasome-dependent[END_REF], enhanced intracellular OPN by about 4 fold. In addition, in the presence of OxLDL, the Piinduced augmentation of OPN was inhibited in a dose-dependent manner. Concerning RANKL, we were unable to determine its intracellular level, but extracellular RANKL level could be assessed after a 5 fold concentration of the culture medium. It was found that OxLDL also prevented Pi-induced augmentation in extracellular RANKL in a dose-dependent manner (Fig 2B)OxLDL impaired Pi signaling via ERK/JNK and AP1/CREB. The next experiment was designed to investigate the effects of OxLDL on Pi signaling. It was found that a 4h preincubation of osteoblasts with 10-50 µg/ml OxLDL enhanced ERK and A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT JNK phosphorylation (Fig 3). Pi induced a 2.3 and 1.6 fold increase in ERK and JNK phosphorylation respectively, and finally, OxLDL inhibited Pi-induced phosphorylation of both kinases. OxLDL also induced an increase in AP1 binding activity, but had no effect on CREB (Fig 4). Pi 4 mM enhanced AP1 and CREB activity by 2.8 and 1.8 fold respectively and a pretreatment with OxLDL prevented this effect, and thus in a dose-dependent manner. OxLDL enhanced the oxidative stress initiated by Pi. In the next experiment, we determined the intracellular level of ROS by means of the fluorescent probe dichloro-fluorescein under Pi and OxLDL treatment. The results from Fig 5

  UMR rat osteoblasts (Fig 1A). OxLDL also prevented Pi-induced expression of Cbfa1/Runx2 transcription factor (Fig 1B), and decreased Pi-induced expression of two osteoblastic markers, OPN and RANKL (Fig 2)

Figure 4 : 16 Figure 5 .

 4165 Figure 4: OxLDL impaired Pi signaling via AP1 and CREB. UMR106 cells in 6 wells-plates were preincubated in DMEM supplemented with 0.1% bovine albumin for down regulation of signalling pathways, 10-50 µg OxLDL protein/ml were added for 4h before a further incubation with 4 mM Pi during 45 min. AP1/CREB DNA binding activity was determined with ELISA kits from Active Motif. Results (OD at 450 nm) are expressed in % of control. Means of 3 independent experiments ± SD. *** p<0.001 by Student's t test.

Figure 6 .

 6 Figure 6. The lipid extract (LE) of OxLDL reproduced whereas Vitamin E prevented the effects of OxLDL. 6a) Reactive oxygen species 6b) Calcium deposition 6c) Intracellular osteopontin. In 6a, the cells were preincubated in medium supplemented with 0.1% albumin and 10 µM vitamin E for 24h before addition of 50 µg OxLDL protein/ml or its lipid extract for 4h. After addition of 4 mM Pi for 45 min, determination of ROS was performed with the fluorescent probe chloro-methyl-2'7'dichlorofluorescein. In 6b-c, cells were incubated in medium supplemented with 5% foetal calf serum, 4mM Pi, 50 µg OxLDL protein/ml or its lipid extract (LE) and 10 µM vitamin E for 8 days (6b) or 4 days (6c). Means of 3 independent experiments ± SD.*: p<0.05; **: p<0.01 and ***: p<0.001 by Student's t test.

Fig. 1 A 22 Fig. 6

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Acknowledgments

We thank C. Louandre and M.A. Conte for skillful technical assistance. This study was supported by INSERM and the University of Picardie-Jules Verne.

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Jan Albrecht

Magdalena Zielińska

Michael D Norenberg

GLUTAMINE AS A MEDIATOR OF AMMONIA NEUROTOXICITY: A CRITICAL APPRAISAL

Keywords: Hepatic encephalopathy, Ammonia, Glutamine, "Trojan Horse" hypothesis, Astrocytes, Mitochondria, Microglia, NO/cGMP pathway

Ammonia is a major neurotoxin implicated in hepatic encephalopathy (HE). Here we discuss evidence that many aspects of ammonia toxicity in HE-affected brain are mediated by glutamine (Gln), synthesized in excess from ammonia and glutamate by glutamine synthetase (GS), an astrocytic enzyme. The degree to which Gln is increased in brains of patients with HE was found to positively correlate with the grade of HE. In animals with HE, a GS inhibitor, methionine sulfoximine (MSO), reversed a spectrum of manifestations of ammonia toxicity, including brain edema and increased intracranial pressure, even though MSO itself increased brain ammonia levels. MSO inhibited, while incubation with Gln reproduced the oxidative stress and cell swelling observed in ammonia-exposed cultured astrocytes. Recent studies have shown that astrocytes swell subsequent to Gln transport into mitochondria and its degradation back to ammonia, which then generates reactive oxygen species and the mitochondrial permeability transition. This sequence of events led to the formulation of the "Trojan Horse" hypothesis. Further verification of the role of Gln in the pathogenesis of HE will have to account for: 1) modification of the effects of Gln by interaction of astrocytes with other CNS cells; and 2) direct effects of Gln on these cells. Recent studies have demonstrated a "Trojan Horse"-like effect of Gln in microglia, as well as an interference by Gln with the activation of the NMDA/NO/cGMP pathway by ammonia as measured in whole brain, a process that likely also involves neurons.

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Introduction

Hepatic encephalopathy (HE) is a complex neuropsychiatric disorder associated with liver dysfunction. The cellular and molecular mechanisms underlying HE are complex, but there is a consensus that an excessive accumulation of ammonia in brain is a primary causative factor (for reviews, see [START_REF] Albrecht | Hepatic encephalopathy: molecular mechanisms underlying the clinical syndrome[END_REF][START_REF] Felipo | Neurobiology of ammonia[END_REF]). Ammonia mainly affects the metabolism and function of astrocytes, leading to impaired astrocytic-neuronal interactions and the ensuing imbalance of neural transmission [START_REF] Norenberg | Astrocytic-ammonia interactions in hepatic encephalopathy[END_REF]. Ammonia induces astrocytic swelling [START_REF] Norenberg | Ammonia-induced astrocyte swelling in primary culture[END_REF] which is the primary cause of cerebral edema, resulting in increased intracranial pressure and brain herniation, a sequence of events often leading to death in patients with HE [START_REF] Blei | Brain edema in acute liver failure: can it be prevented? Can it be treated?[END_REF]. Increase of blood ammonia level, along with EEG abnormalities and impaired psychometric scores, are among key parameters used in the clinical grading of patients with HE [START_REF] Conn | Hepatic encephalopathy: Syndromes and therapies[END_REF].

Since in brain ammonia is initially metabolized to glutamine (Gln), it is not unexpected that HE in humans and experimental animals is usually associated with increased brain and cerebrospinal fluid Gln content [START_REF] Hourani | Cerebrospinal fluid glutamine as a measure of hepatic encephalopathy[END_REF][START_REF] Swain | Ammonia and related amino acids in the pathogenesis of brain edema in acute ischemic liver failure in rats[END_REF][START_REF] Cordoba | Glutamine, myo-inositol, and organic brain osmolytes after portocaval anastomosis in the rat: implications for ammonia-induced brain edema[END_REF]. This increase is particularly pronounced in acute hyperammonemic conditions where brain Gln levels reaches 10 mM concentration in the extracellular space in comatose patients with HE; i.e., a ~20-fold increase above the control levels [START_REF] Tofteng | Persistent arterial hyperammonemia increases the concentration of glutamine and alanine in the brain and correlates with intracranial pressure in patients with fulminant hepatic failure[END_REF]. Gln is an essential precursor of the neurotransmitter amino acids glutamate and GABA, as well as an important energy metabolite in brain [for review, see 11].

For decades the formation of Gln was generally viewed as the principal means of ammonia detoxification. However, in recent years a more sinister aspect of Gln was uncovered; namely, that when synthesized in excess in brain it appears to mediate key aspects of ammonia neurotoxicity. Here we provide supportive evidence in favor of this view. We focus on astrocytic mitochondria as a primary target of Gln, but also hypothesize on the potential role of Gln fluxes to other compartments of the CNS, and speculate how ammonia could affect critical steps in these fluxes. We also discuss recent data indicating that Gln may contribute to ammonia toxicity in other neural cells, e.g., neurons and microglia.

Glutamine as a mediator of ammonia neurotoxicity: experimental demonstration of the phenomenon.

In the pioneering study of Warren and Schenker [START_REF] Warren | Effect of an inhibitor of glutamine synthesis (methionine sulfoximine) on ammonia toxicity and metabolism[END_REF], the glutamine synthetase inhibitor methionine sulfoximine (MSO) was shown to increase the survival of rats acutely exposed to toxic concentrations of ammonia. Unexpectedly, the beneficial effect of MSO became apparent despite the concomitant increase of blood and/or brain ammonia. In hyperammonemic rats or in rats with HE, MSO reversed the biochemical manifestations of ammonia toxicity such as depressed glucose consumption, increased blood-brain barrier transport of aromatic amino acids and other abnormalities (Table 1). MSO administration also attenuated ammonia-induced brain swelling, as manifested by a return of the brain water content to normal (Table 1). In the aforementioned studies, MSO not only decreased cerebral Gln content but it also substantially raised blood and brain ammonia levels. At the cellular level, MSO reduced swelling of astrocytes and pericytes in vivo [START_REF] Koehler | Inhibition of glutamine synthetase reduces ammonia-induced astrocyte swelling in rat[END_REF][START_REF] Tanigami | Effect of glutamine synthetase inhibition on astrocyte swelling and altered astroglial protein expression during hyperammonemia in rats[END_REF], and decreased ammonia-induced swelling of cortical astrocytes in culture [START_REF] Bender | Effects of ammonia on L-glutamate uptake in cultured astrocytes[END_REF]. Pathophysiological manifestations of HE, including an increase of cerebral blood flow and intracranial pressure [START_REF] Master | Cerebral blood flow and the development of ammoniainduced brain edema in rats after portacaval anastomosis[END_REF], excessive potassium accumulation in the intracellular space [START_REF] Sugimoto | Methionine sulfoximine, a glutamine synthetase inhibitor, attenuates increased extracellular potassium activity during acute hyperammonemia[END_REF], and impaired vascular reactivity to CO 2 [START_REF] Takahashi | Restoration of cerebrovascular CO 2 responsivity by glutamine synthesis inhibition in hyperammonemic rats[END_REF] were also corrected by MSO.

Brain Gln content correlates with advancement of symptoms in patients with HE

The development of the NMR technique led in 1990 to the first demonstration of increased Gln content in the brain of a symptomatic HE patient [START_REF] Kreis | Diagnosis of hepatic encephalopathy by proton magnetic resonance spectroscopy[END_REF]. It was later demonstrated that the cerebral Gln content positively correlated with the HE grade [START_REF] Laubenberger | Proton magnetic resonance spectroscopy of the brain in symptomatic and asymptomatic patients with liver cirrhosis[END_REF]. More recently, a bedside brain microdialysis study revealed that in patients with acute HE awaiting liver transplantation due to cirrhosis, cerebral (microdialysate) Gln correlated well with blood ammonia, and with the increased intracranial pressure observed in these patients [START_REF] Tofteng | Persistent arterial hyperammonemia increases the concentration of glutamine and alanine in the brain and correlates with intracranial pressure in patients with fulminant hepatic failure[END_REF].

Altogether, these findings strongly implicate Gln in the pathogenesis of HE.

Toxic effects of glutamine in the CNS are triggered by its accumulation in astrocytic mitochondria

In 1986 Brusilow and Traystman [START_REF] Brusilow | Letter to the editor[END_REF] proposed an interesting hypothesis regarding the potential role of Gln in the mechanism of brain edema in HE/hyperammonemia. These investigators posited that by acting as an osmolyte Gln caused an increase in osmotic pressure in astrocytes which resulted in their swelling. This interpretation assumed the predominant localization of the newly synthesized Gln in astrocytes. The authors considered that as astrocytes account for ~30% of cortical volume, a concentration as high as 30 mM of Gln could be reached in these cells. While an attractive possibility, the available data, however, make this concept difficult to accept. First, Gln accounts for not more than 1.5% of the sum of osmolytes (ions plus small-and medium size soluble molecules) residing in the brain [START_REF] Pasantes-Morales | Mechanisms counteracting swelling in brain cells during hyponatremia[END_REF].

Second, a significant proportion of newly synthesized Gln rapidly leaves astrocytes, both by diffusion and by specific Gln transporters (mainly the N-system transporter SNAT3 [START_REF] Chaudhry | Molecular analysis of system N suggests novel physiological roles in nitrogen metabolism and synaptic transmission[END_REF] which reduces Gln overload in the cells. Furthermore, an increase of brain Gln during HE is accompanied by loss of different low MW osmolytes, including myo-inositol, taurine and betaine [START_REF] Cordoba | Glutamine, myo-inositol, and organic brain osmolytes after portocaval anastomosis in the rat: implications for ammonia-induced brain edema[END_REF]. Perhaps the strongest argument against the osmolyte hypothesis comes from a study demonstrating a negative correlation between astrocyte glutamine levels after ammonia treatment and the extent of cell swelling [START_REF] Jayakumar | Oxidative stress and mitogen-activated protein kinase phosphorylation mediate ammonia-induced cell swelling and glutamate uptake inhibition in cultured astrocytes[END_REF]. Regardless, the notion that glutamine plays a key role in the mechanism of brain edema in acute liver failure represents an important concept put forward by Brusilow and colleagues. The current interpretation is that Gln impairs mitochondrial function secondary to its excessive accumulation in mitochondria. In cultured astrocytes, inhibition of Gln synthesis by MSO suppressed ammonia-induced formation of reactive oxygen species (ROS) [START_REF] Murthy | Ammonia-induced production of free radicals in primary cultures of rat astrocytes[END_REF].When added directly to a non-synaptic mitochondrial preparation [START_REF] Ziemińska | Induction of permeability transition and swelling of rat brain mitochondria by glutamine[END_REF] or cultured astrocytes [START_REF] Pichili | Inhibition of glutamine ransport into mitochondria protects astrocytes from ammonia toxicity[END_REF],

Gln induced mitochondrial permeability transition (mPT) and mitochondrial swelling. In either case, this effect was blocked by the mPT inhibitor cyclosporine A (CsA), and by an inhibitor of mitochondrial Gln uptake, histidine. Recently, the finding with histidine was verified in an in vivo setting in that histidine administered i.p. prevented the brain edema in rats with thioacetamide-induced liver failure [START_REF] Rao | Brain edema in acute liver failure: inhibition by L-histidine[END_REF]. In unpublished observations we found that histidine completely blocked i)-the overexpression of astrocytic aquaporin-4 in brains of rats with thioacetamide-induced acute liver failure and ii) -the ammonia-induced activation of NF-kB and MAPKs -factors that are activated by ammonia and whose activation contribute to cell swelling/brain edema [START_REF] Jayakumar | Oxidative stress and mitogen-activated protein kinase phosphorylation mediate ammonia-induced cell swelling and glutamate uptake inhibition in cultured astrocytes[END_REF][START_REF] Sinke | NFkappaB in the mechanism of ammonia-induced astrocyte swelling in culture[END_REF]. Gln added in the absence of ammonia dosedependently increased ROS synthesis in astrocytes, and this effect was suppressed by CsA [START_REF] Rao | Glutamine in the mechanism of ammonia-induced astrocyte swelling[END_REF]. CsA also attenuated Gln-induced swelling of non-synaptic mitochondria [START_REF] Ziemińska | Induction of permeability transition and swelling of rat brain mitochondria by glutamine[END_REF].

In contrast to Gln, ammonia did not induce mPT or mitochondrial swelling indicating that its direct entry to the mitochondria may not be efficient enough to exert a deleterious effect [START_REF] Ziemińska | Induction of permeability transition and swelling of rat brain mitochondria by glutamine[END_REF]. However, Gln uptake to mitochondria was increased in the presence of elevated ammonia levels [START_REF] Dolińska | Ammonia stimulates glutamine uptake to the cerebral non-synaptic mitochondria of the rat[END_REF].This indicates that in hyperammonemic conditions, ammonia accumulating in excess in extramitochondrial cell compartments may potentiate Gln toxicity by facilitating its entry to mitochondria.

Intra-mitochondrial degradation of Gln by phosphate-activated glutaminase (PAG) is a prerequisite for its gliotoxic action: the Trojan Horse hypothesis

Maximum cell volume increase in ammonia-treated astrocytes occurred at a time when A c c e p t e d M a n u s c r i p t their Gln content had already returned to control values [START_REF] Sinke | NFkappaB in the mechanism of ammonia-induced astrocyte swelling in culture[END_REF], implicating a Gln-dependent process (likely ROS and/or the mPT) in the mediation of astrocyte swelling rather than the Gln molecule per se. In support of this mechanism, swelling of ammonia-treated astrocytes was prevented by the glutaminase inhibitor (6-diazo-5-oxo-L-norleucine; DON) [START_REF] Sinke | NFkappaB in the mechanism of ammonia-induced astrocyte swelling in culture[END_REF]. Of note, inhibition of mitochondrial Gln transport by histidine, which ameliorated Gln toxicity in different experimental settings (see above section), specifically involves a Gln pool of an as yet undefined origin and location which is easily accessible to intramitochondrial deamidation [START_REF] Ziemińska | Analysis of glutamine accumulation in rat brain mitochondria in the presence of a glutamine uptake inhibitor, histidine, reveals glutamine pools with a distinct access to deamidation[END_REF]. On the basis of these results, a "Trojan Horse" hypothesis was formulated [START_REF] Albrecht | Glutamine: a Trojan horse in ammonia neurotoxicity[END_REF]; excess Gln accumulating in astrocytic cytoplasm (the Trojan Horse) crosses mitochondrial membrane and unloads in mitochondrial interior excess ammonia. The Gln-derived ammonia triggers mitochondrial impairment, and an ensuing chain of deleterious events leading to astrocytic dysfunction, swelling and brain edema. Current evidence suggests that the tricarboxylic cycle and the malate-aspartate shuttle enzymes are the primary astrogliaspecific mitochondrial targets of ammonia [START_REF] Faff-Michalak | The two catalytic components of the 2-oxoglutarate dehydrogenase complex in rat cerebral synaptic and nonsynaptic mitochondria: comparison of the response to in vitro treatment with ammonia, hyperammonemia, and hepatic encephalopathy[END_REF][START_REF] Faff-Michalak | Aspartate aminotransferase, malate dehydrogenase, and pyruvate carboxylase activities in rat cerebral synaptic and nonsynaptic mitochondria: effects of in vitro treatment with ammonia, hyperammonemia and hepatic encephalopathy[END_REF][START_REF] Hertz | Energy metabolism in brain cells: effects of elevated ammonia concentrations[END_REF][START_REF] Hertz | Neuronal-astrocytic and cytosolic-mitochondrial metabolite trafficking during brain activation, hyperammonemia and energy deprivation[END_REF].

Gln fluxes from astrocytes to other CNS compartments as potential target of ammonia

In situ the availability of Gln for entry to astrocytic mitochondria will not only depend on its excessive accumulation in that organelle, but also upon the rate at which it is cleared from the cells. Only a fraction of newly synthesized Gln will be transported to astrocytic mitochondria as a significant proportion will exit astrocytes and enter neurons, to give rise to the amino acid neurotransmitters glutamate (Glu) and GABA, reflecting one component of the glutamate Glu/Gln cycle [START_REF] Albrecht | Glutamine in the central nervous system: function and dysfunction[END_REF]. The direct effect of ammonia on Gln efflux from astrocytes, or its uptake by neurons, have not as yet been investigated. Of note, MSOthe agent used in the aforementioned studies to inhibit GS, induces a massive efflux of Gln from astrocytes in an in A c c e p t e d M a n u s c r i p t vitro system [START_REF] Albrecht | L-methionine-DL-sulfoximine induces massive efflux of glutamine from cortical astrocytes in primary culture[END_REF]. To what extent this phenomenon affects the interpretation of the Trojan Horse mechanism is unknown.

A certain proportion of astrocyte-derived Gln will leave the CNS via the cerebral capillary endothelial cells forming the blood-brain barrier [START_REF] Lee | Glutamine transport by the blood-brain barrier: a possible mechanism for nitrogen removal[END_REF]. Increased Gln transport has been demonstrated in cerebral microvessels incubated with ammonia [START_REF] Cangiano | Brain microvessels take up large neutral amino acids in exchange for glutamine. Cooperative role of Na+-dependent and Na+-independent systems[END_REF], or microvessels derived from rats with HE [START_REF] Hilgier | Characteristics of large neutral amino acid-induced release of preloaded L-glutamine from rat cerebral capillaries in vitro: effects of ammonia, hepatic encephalopathy, and gamma-glutamyl transpeptidase inhibitors[END_REF]. Increased Gln efflux across the blood-brain barrier could favor the efflux of Gln from astrocytes and thereby alleviate the toxicity of Gln on astrocytic mitochondria.

Gln transport in CNS cells participating in the different components of the Gln/Glu cycle is mediated by multiple carriers, some of which are either cell-specific or dominate in a given cell type. Gln uptake to, and efflux from, astrocytes occurs mainly via the system N carrier, SN1 (SNAT3) [START_REF] Chaudhry | Molecular analysis of system N suggests novel physiological roles in nitrogen metabolism and synaptic transmission[END_REF], whereas Gln uptake to neurons involves the system A carriers SAT1 and SAT2 (SNAT2, SNAT1) [START_REF] Chaudhry | Glutamine uptake by neurons: interaction of protons with system a transporters[END_REF]. Gln transport from brain to blood is mediated by carriers belonging to systems A, N and ASC [START_REF] Lee | Glutamine transport by the blood-brain barrier: a possible mechanism for nitrogen removal[END_REF][START_REF] O'kane | Na + -dependent neutral amino acid transporters A, ASC, and N of the blood-brain barrier: mechanisms for neutral amino acid removal[END_REF]. Effects of ammonia and HE on the expression and/or activity of astrocytic or neuronal Gln transporters has not been investigated as yet. Stimulation of Gln efflux from cerebral capillary endothelial cells occurs in exchange for tryptophan and other large neutral amino acids and as such has been suspected to involve system L [START_REF] Cangiano | Brain microvessels take up large neutral amino acids in exchange for glutamine. Cooperative role of Na+-dependent and Na+-independent systems[END_REF][START_REF] Hilgier | Changes in the extracellular profiles of neuroactive amino acids in the rat striatum at the asymptomatic stage of hepatic failure[END_REF]. However, the effect on the expression of system L isoforms has not been investigated, and the role of system N has not been examined.

The activity of the N system-mediated Gln efflux from astrocytes is controlled by Glu released from neurons [START_REF] Bröer | Astroglial glutamine transport by system N is upregulated by glutamate[END_REF]. Hyperammonemia or HE are invariably associated with increased Glu accumulation in the extracellular space of the CNS [START_REF] Hilgier | Changes in the extracellular profiles of neuroactive amino acids in the rat striatum at the asymptomatic stage of hepatic failure[END_REF][START_REF] Michalak | Neuroactive amino acids and glutamate (NMDA) receptors in frontal cortex of rats with experimental acute liver failure[END_REF][START_REF] Hermenegildo | Activation of N-methyl-D-aspartate receptors in rat brain in vivo following acute ammonia intoxication: characterization by in vivo brain microdialysis[END_REF][START_REF] Zielińska | Ammonia-induced extracellular accumulation of taurine in the rat striatum in vivo: role of ionotropic glutamate receptors[END_REF], an effect related to decreased astrocytic Glu uptake [START_REF] Albrecht | Astrocytes in acute hepatic encephalopathy: Metabolic properties and transport functions in Biochemical Pathology of Astrocytes[END_REF][START_REF] Knecht | Decreased glutamate transporter (GLT-1) expression in frontal cortex of rats with acute liver failure[END_REF][START_REF] Norenberg | The glial glutamate transporter in hyperammonemia and hepatic encephalopathy: relation to energy metabolism and glutamatergic neurotransmission[END_REF]. This increased extracellular Glu may promote increased astrocytic Gln efflux. In favor of this concept, extracellular brain Gln content is often found increased in hyperammonemia or in animal models of HE [START_REF] Hilgier | Changes in the extracellular profiles of neuroactive amino acids in the rat striatum at the asymptomatic stage of hepatic failure[END_REF][START_REF] Vogels | Memantine, a noncompetitive NMDA receptor antagonist improves hyperammonemia-induced encephalopathy and acute hepatic encephalopathy in rats[END_REF] or humans A c c e p t e d M a n u s c r i p t 9 9 [START_REF] Tofteng | Persistent arterial hyperammonemia increases the concentration of glutamine and alanine in the brain and correlates with intracranial pressure in patients with fulminant hepatic failure[END_REF]. Alternatively or additionally, the increase of extracellular Gln may be a direct result of increased GS activity, which is strongly pronounced at the early stages of HE [see 53 for review], and may be promoted by increased extracellular Glu [START_REF] Lehmann | High extracellular glutamate modulates expression of glutamate transporters and glutamine synthetase in cultured astrocytes[END_REF].

While intracellular His is a well documented inhibitor of mitochondrial Gln transport [START_REF] Ziemińska | Induction of permeability transition and swelling of rat brain mitochondria by glutamine[END_REF][START_REF] Albrecht | Modulation of glutamine uptake and phosphate-activated glutaminase activity in rat brain mitochondria by amino acids and their synthetic analogues[END_REF], extracellular His competes with Gln for the cell membrane N system transport in astrocytes [START_REF] Chaudhry | Molecular analysis of system N suggests novel physiological roles in nitrogen metabolism and synaptic transmission[END_REF]. Since in astrocytes the N system carriers mediate both Gln efflux and influx, part of the cerebral edema-ameliorating effect of His during HE [START_REF] Rao | Brain edema in acute liver failure: inhibition by L-histidine[END_REF] could have been due to inhibition of Gln re-entry to astrocytes. The above considerations further substantiate the need to analyze the response of astrocytic Gln carriers, in particular N system carriers, to ammonia exposure in situ and in vitro.

Aspects of ammonia neurotoxicity that appear not to correlate with increased brain

Gln levels

A few studies have suggested that the accumulation of Gln accumulation may not be critical in the development of cerebral edema and other manifestations of acute HE. Close analysis of the evidence indicates that this may not be the case. Two studies explored the utility of mild hypothermia as a treatment modality for the brain edema associated with HE.

The first study found that hypothermia delayed the development of brain edema in portacaval shunted rats infused with ammonia, without similarly correcting the ammonia-induced rise in cerebral glutamine content [START_REF] Cordoba | Glutamine, myo-inositol, and organic brain osmolytes after portocaval anastomosis in the rat: implications for ammonia-induced brain edema[END_REF]. A later study using a combined 1 H and 13 C NMR spectroscopy demonstrated that while the cerebral edema in rats with acute liver failure was associated with an increase in Gln brain content and glucose-derived products (lactate and alanine), the amelioration of the edema by mild hypothermia was associated with reduction in brain alanine and lactate levels, but not Gln levels [START_REF] Scorticati | Hyperammonemia, brain edema and blood-brain barrier alterations in prehepatic portal hypertensive rats and paracetamol intoxication[END_REF]. It must be noted, however, that hypothermia decreases brain metabolism and more importantly, decreases CBF which would impact on the severity of intracranial pressure (it would decrease it) [START_REF] Stravitz | Therapeutic hypothermia for acute liver failure[END_REF]. Also, the Gln concentration provided does not define in what compartment the Gln is located (intra-vs extracellular), nor its distribution within cells of the same type. Hypothermia has many effects on the CNS and its failure to influence Gln levels cannot be taken as evidence that glutamine does not play a pivotal role in the pathogenesis of the brain edema in ALF.

Ammonia-induced swelling of cerebral cortical slices was prevented by NMDA receptor antagonists and scavengers of oxygen and nitrogen free radicals, supporting the role of oxidative/nitrosative stress in brain edema; however, improvement occurred despite the Gln content in the slices remaining elevated [START_REF] Zielińska | Excitotoxic mechanism of cell swelling in rat cerebral cortical slices treated acutely with ammonia[END_REF]. Likewise, (1) Gln may not exclusively reside in astrocytes, and (2) attenuation of oxidative/nitrosative stress (ONS) could have directly decreased energy metabolism, making brain tissue less sensitive to the mitochondrial dysfunction exerted by Gln. Analogous to the effects of hypothermia, reports on the amelioration of ONS are not to be viewed as contradictory to the Gln/Trojan Horse theory.

In ammonium acetate-treated rats, the transition of behavioral manifestations of HE from moderate to precomatous stage (stage III) or to coma (stage IV) was not prevented by inhibition of Gln synthesis with MSO [START_REF] Dombro | Association between cell swelling and glycogen content in cultured astrocytes[END_REF]. The following factors may explain these apparently contradictory findings: (1) Neurobehavioral manifestations of advanced HE develop in part independently of brain edema [START_REF] Scorticati | Hyperammonemia, brain edema and blood-brain barrier alterations in prehepatic portal hypertensive rats and paracetamol intoxication[END_REF]. (2) Instant elevation of ammonia by MSO treatment could have superimposed a component of ammonia toxicity on the pre-existing mitochondrial dysfunction caused by Gln. (3) Impairment of mitochondrial function by Gln at moderate levels of HE could have sensitized the brain to MSO, effects independent of its inhibition of GS. Moreover, MSO is a convulsing agent frequently used to model epileptic discharges in animals [START_REF] Peters | Glutamic acid and glutamine metabolismin cerebral cortex after seizures induced by methionine sulphoximine[END_REF], which at the cellular level is associated with increased glycogen synthesis in astrocytes [START_REF] Cloix | Epilepsy, Regulation of brain energy metabolism and neurotransmission[END_REF], a reaction also directly evoked by astrocytic swelling [START_REF] Dombro | Association between cell swelling and glycogen content in cultured astrocytes[END_REF]. As judged from studies in in vitro systems, MSO inhibits Gln uptake in one or more CNS cell types in brain slices [START_REF] Zielińska | Effects of methionine sulfoximine on the glutamine and glutamate content and cell volume in rat cerebral cortical slices: involvement of mechanisms not related to inhibition of glutamine synthesis[END_REF], and induces a massive Gln efflux from astrocytes [START_REF] Chaudhry | Glutamine uptake by neurons: interaction of protons with system a transporters[END_REF]. Taken together, caution must be exercised when attempting to implicate a role of Gln solely on the basis of the effects of MSO, which under certain experimental conditions may overemphasize Gln-unrelated manifestations of ammonia neurotoxicity. It will be worthwhile to use in the future studies other, potentially less toxic interventions to manipulate GS activity; of these GS knockdown by siRNA would appear to be the method of choice.

Effects of Gln at targets other than astrocytic mitochondria

Attenuation of the NO/cGMP pathway activity.

Alterations in NO and cGMP levels play varied and independent roles in the pathogenesis of HE. In acute HE, increased NO synthesis, often associated with the activation of NMDA receptors, contributes to oxidative/nitrosative stress (reviewed by [START_REF] Norenberg | Oxidative and nitrosative stress in ammonia neurotoxicity[END_REF][START_REF] Norenberg | Oxidative stress in the pathogenesis of hepatic encephalopathy[END_REF], and elevated cGMP level serves as marker of this activation [START_REF] Hermenegildo | Activation of N-methyl-D-aspartate receptors in rat brain in vivo following acute ammonia intoxication: characterization by in vivo brain microdialysis[END_REF][START_REF] Hilgier | A novel glycine site-specific N-methyl-Daspartate receptor antagonist prevents activation of the NMDA/NO/CGMP pathway by ammonia[END_REF]. A recent microdialysis study showed that increasing extracellular Gln in the brain to levels found in patients with acute HE, caused a decrease in NO and cGMP production in both normal and ammonia-exposed brain. This occurred by limiting the transport of the NO precursor, arginine (Arg), to most likely via the hybrid y + LAT2 system for which Gln and Arg compete [START_REF] Hilgier | Glutamine inhibits ammonia-induced accumulation of cGMP in rat striatum limiting arginine supply for NO synthesis[END_REF]. Whether interference of the NO/cGMP pathway by Gln is beneficial or detrimental for the HE-affected brain is unknown.

The implications may differ in different stages of the disease. In acute HE, decreased NO synthesis might ameliorate symptoms by decreasing the severity of oxidative/nitrosative stress, including astrocytic swelling, an event to which protein kinase G activation contributes [START_REF] Konopacka | Protein kinase G is involved in ammoniainduced swelling of astrocytes[END_REF]. Chronic HE is associated with a deficit in cGMP, which may be responsible for cognitive and memory impairments [START_REF] Monfort | Chronic liver failure in rats impairs glutamatergic synaptic transmission and long-term potentiation in hippocampus and learning ability[END_REF]. Whether in chronic HE Gln inhibits cGMP synthesis in neurons remains to be investigated. If it does, it will add a role for Gln in the cGMP-A c c e p t e d M a n u s c r i p t dependent impairment in memory and cognition. Clarification of whether Gln interferes with Arg transport in neurons, astrocytes or both is needed to better understand its pathophysiological impact. The NMDA receptor-mediated activation of the NO/cGMP pathway by ammonia was originally thought to occur mainly, if not exclusively in neurons.

However, evidence indicates that this pathway also occurs in astrocytes [START_REF] Schliess | Ammonia induces MK-801-sensitive nitration and phosphorylation of protein tyrosine residues in rat astrocytes[END_REF], where it likewise contributes to oxidative/nitrosative stress [72, and references therein]. The issue is further complicated by the paucity and inconclusiveness of data regarding y+ LAT2 expression in the different neural cells [START_REF] Bröer | The heterodimeric amino acid transporter 4F2hc/y+LAT2 mediates arginine efflux in exchange with glutamine[END_REF][START_REF] Bae | Y+ and y+ L arginine transporters in neuronal cells expressing tyrosine hydroxylase[END_REF].

Gln as a Trojan Horse in microglia?

A recent study documented that exogenously added Gln causes apoptosis in a microglial cell line and that this effect was prevented upon inhibition of Gln degradation by the PAG inhibitor, DON [START_REF] Svoboda | L-Glutamine-induced apoptosis in microglia is mediated by mitochondrial dysfunction[END_REF]. Also, Gln mimicked ammonia in producing the oxidative stress response in these cells. However, the findings remain to be confirmed with native microglial cells where GS activity has not been definitively demonstrated. These findings also require their validation in an in vivo HE model. More work is needed to interpret the effect observed in the microglial cell line as another pathologic manifestation ammonia leading to the "Trojan Horse" pathway as reported for astrocytes.

Concluding remarks

The available evidence suggests that the most profound effect of excess of Gln accumulating in the CNS during HE is an impairment of mitochondrial function in astrocytes. into glutamine (Gln) in astrocytes (and perhaps microglia) by the amidation of glutamate (Glu) to glutamine (Gln), a process catalyzed by glutamine synthase (GS). Gln accumulated in astrocytes i) crosses the mitochondrial membrane and is hydrolyzed by phosphate-activated glutaminase (PAG) to glutamate (Glu) and NH4 + , and ii) exits astrocytes preferably by the system N (the SNAT3 transporter) which is controlled by extracellular Glu, and partly by system L in exchange for large neutral amino acids (NAA). The Gln that leaves astrocytes enters neurons mainly via system A to be converted to the amino acid neurotransmitters Glu and GABA, and possibly by system y + L (the y + LAT2 transporter) in exchange of Arg, which serves the purpose of modulating the availability of Arg for NO synthesis and thus may influence the rate of the NO/cGMP pathway. A portion of Gln derived from astrocytes leaves the CNS via the L and N systems that are localized in capillary endothelial cells that forms the blood-brain barrier, some of the Gln may enter the microglial cells and modulate their function.

A c c e p t e d M a n u s c r i p t

  This results in the intramitochondrial liberation of ammonia, induction of the mPT and the generation of free radicals resulting in astrocytic swelling and brain edema. Deregulation of the NO/cGMP pathway by Gln due to interference with Arg transport across the cell membrane and the promotion of microglial apoptosis represent potential new factors in the A c c e p t e d M a n u s c r i p t 13 13 mechanism of HE, but whose physiological significance requires further investigation. The actions of Gln at different targets are likely to be operative in the pathogenesis of HE, and this in turn may be complicated by the effects of ammonia or Gln itself on the transfer of newly synthesized astrocytic Gln to other compartments of the CNS (Fig.1). Influencing the synthesis of glutamine, its metabolism, and intra-extracellular transport may yield promising novel therapeutic approaches for HE and other hyperammonemic states.

Fig. 1 .

 1 Fig. 1. Major steps of Gln metabolism and transport in the CNS which may contribute to, or modulate, Gln-mediated ammonia toxicity. Ammonia (NH 4 + ) is incorporated ("detoxified")

A c c e p t e d M a n u s c r i p

Figure 1 A c c e p t e d M a n u s c r i p

 1 Figure 1

Figure

  Figure

Table 1 .

 1 Effects of MSO on the major HE parameters in hyperammonemic (HA) or portacaval shunted (PCS) rats.1 ref.[START_REF] Takahashi | Restoration of cerebrovascular CO 2 responsivity by glutamine synthesis inhibition in hyperammonemic rats[END_REF]; 2 ref.[START_REF] Hawkins | Effect of reducing brain glutamine synthesis on metabolic symptoms of hepatic encephalopathy[END_REF].Values significantly different from respective controls (p<0.05 by different tests) are in bold.

		Control	HA	PCS	HA + MSO	PCS+MSO
	Ammonia (blood)
	(μM) 1	29 ±3 1	601 ± 38 1		908 ± 196 1
	(nmol/l) 2	97 ± 8 2		227 ± 15 2		265 ± 27 2
	Ammonia (brain)
	(nmol/g) 1	146 ± 6 1		384 ± 22 1		424 ± 46 1
	GS (brain) (IU/g) 1	38.8 ± 0.8 1	39.9 ± 0.3 1		14.1 ± 0.2 1
	Gln (brain)
	(mmol/kg) 1	5.6 ± 0.4 1	18.8 ± 0.4 1	8780 ± 570 2	2.6 ± 0.4 1	5180 ± 540 2
	(nmol/g) 2	5580 ± 360 2
	Glu (brain)
	(mmol/kg) 1	10.1 ± 1.0 1	6.6 ± 0.4 1		9.5 ± 0.6 1
	Spec. gravity	1.0452 1	1.0424 1		1.0446 1
	(brain) 1
	Water content	0.783 1	0.804 1		0.784 1
	(brain) (%) 1
	Tryptophan (brain)
	(nmol/g) 2	17 ± 1 2		45 ± 3 2		25 ± 2 2
	Glucose
	consumption (brain)	861 ± 53 2		614 ± 39 2		733 ± 87 2
	(nmol/min/g) 2
	BBB transport of
	tryptophan	18 ± 2 2		31 ± 5 2		15 ± 3 2
	(μl/min/g) 2

Acknowledgments: Supported by the Ministry of Science and Education of Poland, grants no S 005/P-N/2007/01 (JA) and NN 401 0550 33 (MZ), and by a Department of Veterans Affairs Merit Review Award and NIH grant DK06331 (MDN).

Ammonia (NH 4 + ) is incorporated ("detoxified") into glutamine (Gln) in astrocytes (and perhaps microglia) by the amidation of glutamate (Glu) to glutamine (Gln), a process catalyzed by glutamine synthase (GS). Gln accumulated in astrocytes i) crosses the mitochondrial membrane and is hydrolyzed by phosphate-activated glutaminase (PAG) to glutamate (Glu) and NH4 + , and ii) exits astrocytes preferably by the transport system N (the SNAT3 transporter), and partly by system L in exchange for large neutral amino acids. The Gln that leaves astrocytes enters neurons mainly via system A to be converted to Glu, and possibly by system y + L (the y + LAT2 transporter) in exchange of Arg, which serves the purpose of modulating the availability of Arg for NO synthesis and thus may influence the rate of the NO/cGMP pathway. A portion of Gln derived from astrocytes leaves the CNS via the L and N systems that are localized in the cerebral capillary endothelial cells that forms the blood-brain barrier, portion may be enter the microglial cells and modulate their function. An extended version of the figure appeared as Fig. 1. in the manuscript.

*Graphical Abstract