Iulia Mocan

Florian Georgescauld

Philippe Gonin

Didier Thoraval

Laura Cervoni

Anna Giartosio

Sanbrine Dabernat-Arnaud

Marc Crouzet

Marie-Lise Lacombe

Ioan Lascu
email: ioan.lascu@ibgc.u-bordeaux2.fr

Sandrine Dabernat-Arnaud #

Protein phosphorylation corrects the folding defect of the neuroblastoma

Keywords: NDP, nucleoside diphosphate, ANS, 8-anilino-1naphthalene sulfonate, BisANS, 4 Nucleoside diphosphate kinase, nm23, histidine phosphorylation, neuroblastoma, folding intermediate, molten globule

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Nucleoside diphosphate (NDP 1 ) kinases catalyze the reversible transfer of the γ-phosphoryl group of nucleoside triphosphates to nucleoside diphosphates [START_REF] Janin | Nucleoside-diphosphate kinase: structural and kinetic analysis of reaction pathway and phosphohistidine intermediate[END_REF][START_REF] Lascu | The catalytic mechanism of nucleoside diphosphate kinases[END_REF]. The two-step reaction has a covalent intermediate that is transiently phosphorylated on the histidine residue 118 in human NDP kinase A [START_REF] Gilles | Nucleoside diphosphate kinase from human erythrocytes. Structural characterization of the two polypeptide chains responsible for heterogeneity of the hexameric enzyme[END_REF][START_REF] Morera | Mechanism of phosphate transfer by nucleoside diphosphate kinase: X-ray structures of the phosphohistidine intermediate of the enzymes from Drosophila and Dictyostelium[END_REF]: ------------------------------------------------------- NDP kinases have a single nucleotide binding site per subunit, where the NTP and the NDP bind sequentially during the catalytic cycle (reactions 1a and 1b).

Mg
In addition to their catalytic function, eukaryotic NDP kinases are involved in complex regulatory functions, some of which are not associated with kinase activity. Drosophila NDP kinase, the product of the awd gene, is essential for larvae development [START_REF] Timmons | Role of AWD/nucleoside diphosphate kinase in Drosophila development[END_REF]. Human NDP kinase B, also called Nm23-H2, is a transcription factor for the proto-oncogene c-myc [START_REF] Postel | Multiple biochemical activities of NM23/NDP kinase in gene regulation[END_REF] and has nuclease activity [START_REF] Ma | The metastasis suppressor NM23-H1 possesses 3'-5' exonuclease activity[END_REF][START_REF] Fan | Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTL-mediated apoptosis, and the nucleosome assembly protein SET is its inhibitor[END_REF].

Crosslinking experiments demonstrate that both NDP kinases A and B bind to DNA in vivo [START_REF] Cervoni | In vivo cross-linking of nm23/nucleoside diphosphate kinase to the PDGF-A gene promoter[END_REF].

NDP kinase A (Nm23-H1) is an antimetastatic protein [START_REF] Steeg | Metastasis suppressor genes: basic biology and potential clinical use[END_REF]. Low expression is associated with high metastatic potential in tumors and cells in culture. Overexpression of the protein in highly metastatic cell lines decreases their metastatic potential [START_REF] Leone | Reduced tumor incidence, metastatic potential, and cytokine responsiveness of nm23transfected melanoma cells[END_REF]. The mechanism of NDP kinase A action in metastasis progression is still not well understood. Recently, NDP kinase A was reported to act in a coordinated manner with the human equivalent of the Drosophila prune protein in the control of cancer metastasis [START_REF] D'angelo | Prune cAMP phosphodiesterase binds nm23-H1 and promotes cancer metastasis[END_REF].

The S120G mutation in NDP kinase A has been found in several high-grade neuroblastomas, but not in low-grade tumors or in control tissues [START_REF] Chang | Nm23-H1 mutation in neuroblastoma[END_REF]. This suggests that the presence of the mutant protein my be associated with tumor aggressiveness. Indeed, the mutant appears to promote neuroblastoma more effectively than the wild-type protein when expressed in an NB69-derived human neuroblastoma cell line [START_REF] Almgren | Nucleoside diphosphate kinase A/nm23-H1 promotes metastasis of NB69-derived human neuroblastoma[END_REF]. If the S120G mutation is transfected into human breast carcinoma cell lines, the resulting mutant NDP kinase no longer inhibits motility and if the mutation is transfected into prostate cancer cells the mutant NDP kinase no longer inhibits colonization and invasion. [START_REF] Kim | Point mutations affecting the oligomeric structure of Nm23-H1 abrogates its inhibitory activity on colonization and invasion of prostate cancer cells[END_REF]. The S120G mutant has impaired affinity for the human prune protein [START_REF] Forus | Amplification and overexpression of PRUNE in human sarcomas and breast carcinomas-a possible mechanism for altering the nm23-H1 activity[END_REF]. Wild-type NDP kinase A reduces the acute desensitization of muscarinic K + channels, whereas the S120G mutant kinase suppresses this effect and even accelerates desensitization [START_REF] Otero | Wild-type NM23-H1, but not its S120 mutants, suppresses desensitization of muscarinic potassium current[END_REF]. Copyright 2006 Biochemical Society

Despite the proximity of the serine 120 to the active site histidine and its conservation in all known NDP kinases, the S120G mutation only slightly decreases the kinase activity [START_REF] Lascu | A point mutation of human nucleoside diphosphate kinase A found in aggressive neuroblastoma affects protein folding[END_REF][START_REF] Schaertl | Human nucleoside diphosphate kinase B (Nm23-H2) from melanoma cells shows altered phosphoryl transfer activity due to the S122P mutation[END_REF]. It decreases the stability of the protein to denaturation [START_REF] Lascu | A point mutation of human nucleoside diphosphate kinase A found in aggressive neuroblastoma affects protein folding[END_REF][START_REF] Chang | A nucleoside diphosphate kinase A (nm23-H1) serine 120-->glycine substitution in advanced stage neuroblastoma affects enzyme stability and alters protein-protein interaction[END_REF]. A folding defect is the most substantial effect of the mutation: the urea-denatured S120G mutant cannot refold in vitro [START_REF] Lascu | A point mutation of human nucleoside diphosphate kinase A found in aggressive neuroblastoma affects protein folding[END_REF]. Instead, it accumulates as a "molten globule" folding intermediate, as assessed by various biochemical and biophysical techniques. The wild-type enzyme refolds under the same conditions and associates to form active hexamers.

To understand the structural basis of the folding defect of the mutant S120G, its X-ray structure has been solved in our Laboratory [START_REF] Giraud | Crystal structures of S120G mutant and wild type of human Nucleoside Diphosphate Kinase A in complex with ADP[END_REF]. The mutation does not affect subunit conformation or subunit assembly. The mutant and wild-type hexamers are identical within the experimental error (r.m.s. on C α positions of 0.3 Å). Thus, incorporation of mutant subunits into hexamers probably does not affect other subunits. Heterohexamer formation is a common property of hexameric NDP kinases [22]. The S120G mutation is probably not dominant negative in the sense that S120G mutant subunits will not change the function of the other subunits in the hexamer. S120G mutant subunits and H118N mutant subunits has been shown to associate to form hexamers in vitro and are active 2

However, the S120G mutation may seem to be "dominant" if a folding intermediate or lower order oligomers are present in cells with the S120G mutant NDP kinase but not in cells with wild-type NDP kinase. A large amount of dimeric S120G mutant protein has been found in vitro by crosslinking [START_REF] Kim | Point mutations affecting the oligomeric structure of Nm23-H1 abrogates its inhibitory activity on colonization and invasion of prostate cancer cells[END_REF][START_REF] Chang | A nucleoside diphosphate kinase A (nm23-H1) serine 120-->glycine substitution in advanced stage neuroblastoma affects enzyme stability and alters protein-protein interaction[END_REF]. Immunoprecipitation experiments showed that the S120G mutant protein interacts with a 28 kDa protein. This interaction did not occur with the wild-type protein [START_REF] Chang | A nucleoside diphosphate kinase A (nm23-H1) serine 120-->glycine substitution in advanced stage neuroblastoma affects enzyme stability and alters protein-protein interaction[END_REF].

A similar natural mutation is the killer-of-prune mutation (K-pn) in the Drosophila NDP kinase.

The replacement of the proline at position 97 by a serine results in a dominant, conditional and lethal phenotype [START_REF] Biggs | Analysis of the lethal interaction between the prune and Killer of prune mutations of Drosophila[END_REF]. The nature of the particular molecular species which confers the dominant character is still unknown, but biochemical evidence suggests that it is the folded monomer [START_REF] Lascu | A Pro/Ser substitution in nucleoside diphosphate kinase of Drosophila melanogaster (mutation Killer of prune) affects stability but not catalytic efficiency of the enzyme[END_REF].

The S120G mutant NDP kinase A folds and associates into an active hexameric protein in mammalian cells and E. coli but cannot do this in vitro. We discovered that phosphorylation of the active-site histidine, either enzymatic or chemical with phosphoramidate, corrects the folding defect of the S120G mutant. In agreement with the proposed mechanism, the folding defect persists if the active-site histidine, phosphorylated during the catalytic cycle, is replaced by an asparagine in the S120G protein.

EXPERIMENTAL PROCEDURES

Mutagenesis and protein purification. S120G and H118N mutant constructs were generated using the Transformer TM site-directed mutagenesis kit (Clonetech). The mutagenesis was performed on the PET21b (Novagen) construct to express the NDP kinase A. The mutagenic primers designed to produce the point mutations were 5'-CATTATACATGGCGGTGATTCTGTGG-3' and 5'-AGGAACATTATAAATGGCAGTGATTCT-3' for S120G and H118N, respectively (the altered nucleotides are in bold face). The H118N/S120G double mutant in the pET21nm23 plasmids was made with the Quick Change site-directed mutagenesis kit (Stratagene) following the manufacturer's instructions. Mutations were confirmed by restriction analysis and nucleotide sequencing.

All proteins were expressed using a pET-21 vector. Wild-type protein and S120G and H118N

mutants were purified at 4 °C and analyzed as described previously [START_REF] Lascu | A point mutation of human nucleoside diphosphate kinase A found in aggressive neuroblastoma affects protein folding[END_REF], with the following modifications. The DNase-treated bacterial extract was applied to a QAE-Sepharose column equilibrated with 50 mM Tris-HCl buffer (pH 8.0) containing 2 mM DTT. The enzyme was eluted with a linear gradient of 0 to 1 M NaCl in the same buffer. Fractions containing the H118N protein were identified by electrophoresis and by measurement of activity of the S120 and wild-type proteins. The pH of the fractions containing the enzyme was corrected to 7.0, and the samples were diluted three-fold with distilled water to decrease the ionic strength. Protein phosphorylation. A. Enzymatic. NDP kinase was phosphorylated as described previously [START_REF] Lascu | Pig heart nucleosidediphosphate kinase. Phosphorylation and interaction with Cibacron blue 3GA[END_REF]. The equilibrium constant of phosphorylation reaction 1a is low (see Introduction) Protein phosphorylation. B. Chemical. NDP kinase is phosphorylated on the active-site histidine by phosphoramidate [START_REF] Janin | Nucleoside-diphosphate kinase: structural and kinetic analysis of reaction pathway and phosphohistidine intermediate[END_REF][START_REF] Morera | Mechanism of phosphate transfer by nucleoside diphosphate kinase: X-ray structures of the phosphohistidine intermediate of the enzymes from Drosophila and Dictyostelium[END_REF]. This reagent is not commercially available. The dipotassium salt of phosphoramidate was synthesized as described previously [START_REF] Wei | Identification of phosphohistidine in proteins and purification of protein-histidine kinases[END_REF]. Chemical phosphorylation was achieved with 0.16 M phosphoramidate in 0.16 M Tris-HCl (pH 8.0) and 10 mM MgSO 4 for 30 min.

After denaturation, the NDP kinase was reactivated gel filtration as above.

Analytical techniques Protein concentration was determined from the optical density at 280 nm using an extinction coefficient of 1.35 for 1 mg/ml, which was calculated from the amino acid composition [START_REF] Gill | Calculation of protein extinction coefficients from amino acid sequence data[END_REF]. Enzymatic activity was assayed by a spectrophotometric assay, with 1 mM ATP and 0.2 mM 8-bromoinosine-5'-diphosphate as substrates [START_REF] Lascu | Pig heart nucleosidediphosphate kinase. Phosphorylation and interaction with Cibacron blue 3GA[END_REF]. A LS50B spectrofluorimeter (Perkin Elmer) was used to measure intrinsic protein fluorescence intensity (excitation at 295 nm, emission at 340 nm) and BisANS fluorescence intensity (excitation at 390 nm, emission at 480 nm).

Analytical size-exclusion chromatography was performed using a Superdex 75 column (Pharmacia, Uppsala) equilibrated with 100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM MgCl 2 and 1 mM DTT, in the absence or presence of 100 µM ATP or ADP, at a flow rate of 0.5 ml/min.

The column was calibrated with BioRad Markers. Protein fluorescence was monitored using a flow cell on the Perkin-Elmer LS50B spectrofluorimeter (excitation at 295 nm, bandwidth of 5 nm; emission at 340 nm, bandwidth of 15 nm). In other experiments, the effluent was mixed just before the flow cell with BisANS at a final concentration of 1 µM. BisANS fluorescence was measured (excitation at 370 nm, bandwidth of 5 nm; emission at 480 nm, bandwidth of 15 nm). Protein peaks containing the folding intermediates were indicated by BisANS binding.

All measurements were performed at 25 °C in 100 mM Tris-HCl (pH 8.0) containing 1 mM DTT.

Figures show typical results of experiments performed at least twice with different enzyme

preparations.

RESULTS AND DISCUSSION

Protein phosphorylation assists folding of the S120G mutant NDP kinase A. We coupled reaction 1a with the pyruvate kinase reaction to obtain full protein autophosphorylation.

Phosphorylation was continuously monitored spectrophotometrically with the lactate dehydrogenase reaction. A decrease in NADH reflected the generation of ADP by reaction 1a. We calculated a stoichiometry of 0.85 phosphoryl groups incorporated for each NDP kinase subunit from the data generated by this spectrophotometric analysis (Fig. 1A). The H118N mutant could not be phosphorylated, as expected. The final drift was caused by nonenzymatic hydrolysis of the Spontaneous dephosphorylation is a substantial limitation for protein stability studies. Equilibrium experiments could not be done with the phosphorylated enzyme because most of the phosphohistidine was hydrolysed during incubation. We denatured the phosphorylated NDP kinase and then passed the sample over a HiTrap desalting column to quickly (in about one min) eliminate the denaturant, initiating kinase refolding. The phosphorylated NDP kinase recovered about 60% of its enzymatic activity (Fig. 1B). Very little reactivation has been noted in similar experiments with unphosphorylated enzyme and with enzyme that was dephosphorylated with equilibration buffer containing 100 µM ADP in the in the desalting experiment (Fig. 1B).

The NDP kinases may be phosphorylated on the active-site histidine chemically, with phosphoramidate [START_REF] Janin | Nucleoside-diphosphate kinase: structural and kinetic analysis of reaction pathway and phosphohistidine intermediate[END_REF][START_REF] Morera | Mechanism of phosphate transfer by nucleoside diphosphate kinase: X-ray structures of the phosphohistidine intermediate of the enzymes from Drosophila and Dictyostelium[END_REF]. The crystal structure of the Drosophila and Dictyostelium NDP kinases, phosphorylated in this way in the native state, showed that the other protein histidine residues were not phosphorylated. The S120G mutant was phosphorylated, unfolded and refolding started by gel filtration (Fig. 1B). The reactivation was in quite a good yield, although lower than with the autophosphorylated enzyme. The effect was lost when the chemically phosphorylated enzyme was dephosphorylated by ADP. This result clearly indicate that the active-site phosphorylation assisted the protein folding.

In further renaturation experiments we used unphosphorylated enzyme with MgATP present during enzyme refolding assay. It should be mentioned that the two procedures are not equivalent. In the experiment shown in Fig. 1B, most of the protein remained phosphorylated in the unfolded state.

Thus, the phosphoryl group may assist folding from the initial stages of this process. In contrast, if the unfolded and unphosphorylated kinase is diluted in MgATP-containing buffer, phosphorylation may occur after the formation of a catalytically competent form of the protein, presumably the correctly folded monomer. Refolding in the presence of MgATP is certainly more similar to what occurs in the cellular environment. The active-site histidine can be dephosphorylated by ADP. This demonstrates that the phosphorylated residue is the active-site histidine.

Effect of nucleotides on the reactivation of the S120G mutant NDP kinase A.

Full enzymatic activity of hexameric NDP kinases is associated with the correct quaternary structure. Folded NDP kinase A monomers are catalytically competent, but have a low specific activity that is about 1.5 -2% of that of the hexamer 2 . Similar results have been obtained with the Dictyostelium cytosolic NDP kinase [START_REF] Lascu | Equilibrium dissociation and unfolding of nucleoside diphosphate kinase from Dictyostelium discoideum[END_REF]. Therefore, enzymatic activity measurements indicated the correct folding and association. The urea-unfolded S120G mutant diluted with buffer recovered less than 3% of the control activity in the absence of ATP. The wild-type enzyme recovered 50% of the activity under the same conditions (Fig. 2A). In the presence of ATP and 5 mM MgCl 2, a large amount of mutant kinase that was reactivated (Fig. 2B). ATP and ADP form complexes with Mg 2+ in the presence of [START_REF] Schneider | The mechanism of phosphorylation of anti-HIV D4T by nucleoside diphosphate kinase[END_REF]. Our study shows that ADP was not an activator and that it strongly inhibited the activating effect of MgATP. The nonhydrolysable ATP analog AMP-PNP, shown to bind to NDP kinases with an affinity similar to that of ADP [START_REF] Cervoni | Binding of nucleotides to nucleoside diphosphate kinase: a calorimetric study[END_REF], did not reactivate the NDP kinase (Fig. 2A). It was a weaker inhibitor than ATP in the reactivation assay (Fig. 2B). Thus, NDP kinase is probably reactivated by phosphorylation (Fig. 2B).

The absence of an effect of ADP may seem surprising because its binding to NDP kinases is known to increase their thermodynamic stability. The probable explanation is the following. The binding of ADP and other nucleotides stabilizes the hexamer, whereas phosphorylation probably corrects the folding defect of the S120G mutant by stabilizing the native monomer (see below). The folding intermediate accumulating in the absence of MgATP slowly changes so that it cannot be reactivated. We found that reactivation was fast if the urea-denatured protein was diluted in MgATP-containing buffer. However, if the denatured protein was incubated in the absence of nucleotide before adding MgATP, the reactivation rate was lower (Fig. 2C).

Effect of MgATP on folding/association followed by size-exclusion chromatography.

The sizeexclusion chromatography is useful since the various species present in a protein sample may be separated and identified. In contrast, enzymatic activity measurements detect the enzymatic competent species only, while optical spectroscopic techniques yield mean values of the measured parameter. The native hexamer, folding intermediate and native monomer are well separated by sizeexclusion chromatography. The native hexamer is eluted first at an elution volume of 11.0 ml, then the folding intermediate is eluted at 12.8 ml and finally the native monomer is eluted at 13.8 ml. The elution volumes were reproducible within 0.1 ml. We followed the folding/association pattern in the following manner. We injected the unfolded protein into the column equilibrated either with buffer Phosphorylation of the S120G mutant led to enzyme reactivation by facilitating correct folding and subunit assembly (Fig. 1B). When the unfolded phosphorylated S120G mutant was injected into the column, most of it refolded/assembled on the column and eluted as the hexamer. Some of the protein eluted later at a position characteristic of the folding intermediate. In contrast, if the protein was dephosphorylated by including ADP in the equilibration buffer, all the protein eluted as a folding intermediate (Fig. 3E). Dephosphorylation does occur in this experiment, but after the protein has folded into a catalytically competent form, probably the folded monomer.

Effect of MgATP on the refolding of urea-denatured wild-type and mutant NDP

kinases followed by fluorescence spectroscopy. We next studied the effect of MgATP on the refolding of the NDP kinase A and of its mutants to distinguish between an effect of MgATP on the folding process and subunit association. This is a difficult because the folding intermediate aggregated at the high concentrations necessary for NMR spectroscopy (G. Craescu, unpublished experiments) or for near-UV circular dichroism spectroscopy (not shown). The far-UV circular dichroism spectroscopy is not informative since the folding intermediate accumulated during the refolding of the S120G mutant contains a high percentage of secondary structure [START_REF] Lascu | A point mutation of human nucleoside diphosphate kinase A found in aggressive neuroblastoma affects protein folding[END_REF]. Intrinsic protein fluorescence also was not informative: the folding intermediate and the native protein have emission maximums at 340 nm, but the intermediate has a lower fluorescence intensity than the native protein. The differential binding of BisANS to different molecular forms was the most useful technique in this case. Unfolded wild-type and mutant NDP kinases were diluted in buffer in the presence or absence of MgATP, then BisANS was added immediately (Fig. 4). The renaturing wildtype protein bound less BisANS than the mutants. Only the wild-type and the S120G mutant displayed a decrease in BisANS binding in the presence of MgATP than in its absence. The H118N mutant and the H118N/S120G double mutant were insensitive to the presence of MgATP. Mutation of the active-site histidine abolished phosphorylation, but not ATP binding [START_REF] Schneider | The mechanism of phosphorylation of anti-HIV D4T by nucleoside diphosphate kinase[END_REF]. In all cases, spectra were insensitive to the presence of ADP, or ATP in the absence of MgCl 2 . Even though the H118N mutation is a conservative mutation, it affected protein refolding.

Fig

Finally, we studied the renaturation of NDP kinases in various concentrations of urea, in the absence and presence of MgATP, monitored by BisANS binding (Fig. 5). MgATP did not affect the H118N/S120G double mutant. This is expected if phosphorylation is crucial for folding. In addition, the S120G mutant bound less BisANS in the presence of MgATP at low concentrations of urea, but not in the presence of 2 to 3 M urea. This supports the idea that MgATP promotes the reactivation of the folding-deficient S120G mutant through phosphorylation of the native monomer. A "global" effect would decrease the folding intermediate concentration at all urea concentrations. This has been noted indeed with the natural osmolyte trimethylamine-N-oxide (F. Georgescauld, manuscript in preparation). Phosphoryl transfer may occur only after the formation of the protein active site.

MgATP assisted to some degree the refolding of the H118N mutant which cannot be phosphorylated (Fig. 3).

Fig 5

CONCLUSIONS. BIOLOGICAL SIGNIFICANCE

Our findings suggest the following scheme for the folding/association of NDP kinase A and its mutants (Scheme I). A scheme with the folding intermediate (I) off-pathway and a "triangular" scheme cannot be excluded until kinetic studies are undertaken. The conclusions of this study would not change significantly with these other schemes.

SCHEME 1

The key difference of the human NDP kinase A from other hexameric NDP kinases is that the folding intermediate (I), rather than the folded monomer (M), accumulates during protein refolding.

This is reminiscent to the refolding of Dictyostelium cytosolic NDP kinase at an acidic pH [START_REF] Cervoni | Quaternary structure of Dictyostelium discoideum nucleoside diphosphate kinase counteracts the tendency of monomers to form a molten globule[END_REF]. The The protein being synthesized in vivo on the ribosomes may follow the simple scheme described above. The in vivo [ATP]/[ADP] ratio is high, typically 5 to 10. Under these conditions the unfavorable effect of ADP is small. Once the native M is formed it may be trapped in hexamers with NDP kinase A or B subunits. However, the situation may be more complex. The partially folded mutant protein may be sequestered by chaperone proteins or it may aggregate. Cellular crowding has been shown to favor associations [START_REF] Ellis | Macromolecular crowding: obvious but underappreciated[END_REF]. It could increase the rate of normal hexamer formation and also those of chaperone binding and aggregation. These two side effects may be associated with the observed cellular effects of the S120G mutant NDP kinase A [START_REF] Kim | Point mutations affecting the oligomeric structure of Nm23-H1 abrogates its inhibitory activity on colonization and invasion of prostate cancer cells[END_REF][START_REF] Forus | Amplification and overexpression of PRUNE in human sarcomas and breast carcinomas-a possible mechanism for altering the nm23-H1 activity[END_REF][START_REF] Otero | Wild-type NM23-H1, but not its S120 mutants, suppresses desensitization of muscarinic potassium current[END_REF].

An interesting question is whether the "molten globule" folding intermediate can bind ATP and be autophosphorylated. Our data suggest that the catalytically-competent form is the folded monomer, which agrees with the "preequilibrium" hypothesis [START_REF] James | Conformational diversity and protein evolution--a 60-yearold hypothesis revisited[END_REF]. For example, MgATP did not shift the equilibrium between the folding intermediate and the native monomer in the presence of 2 to 3 M urea (Fig. 5). Various proteins (wild-type and mutant) are misfolded in the absence of substrates, but adopt the native form in their presence [START_REF] Vamvaca | An enzymatic molten globule: efficient coupling of folding and catalysis[END_REF][START_REF] Uversky | Circularly permuted dihydrofolate reductase possesses all the properties of the molten globule state, but can resume functional tertiary structure by interaction with its ligands[END_REF]. In at least one case, the cofactor flavin mononucleotide (FMN), could not interact with intermediate states [START_REF] Bollen | Last in, first out: the role of cofactor binding in flavodoxin folding[END_REF]. In contrast, Copyright 2006 Biochemical Society intermediate [START_REF] Bushmarina | Cofactor effects on the protein folding reaction: acceleration of alpha-lactalbumin refolding by metal ions[END_REF]. One might think that small ligands, including metal ions, can interact with partially or completely unfolded proteins. However, binding of ligands like ATP require a completely formed active site. In addition, the active-site residues must be in the correct position to be catalytically active and for autophophorylation to be possible.

What is the structural basis for the correction of the folding defect by phosphorylation? The X-ray structure of the phosphorylated NDP kinase A is not known. Therefore, we used the highly similar Drosophila NDP kinase structure phosphorylated on the active-site histidine [START_REF] Morera | Mechanism of phosphate transfer by nucleoside diphosphate kinase: X-ray structures of the phosphohistidine intermediate of the enzymes from Drosophila and Dictyostelium[END_REF] as a model to detect interactions in the phosphorylated enzyme (Fig. 6). The side chain of Ser121 forms a hydrogen bond with Glu130, which is part of the α4 helix. The phosphoryl group interacts with Tyr53 which belongs to a different part of the subunit (αA helix). The phosphorylation does not replace the missing interactions resulting from the absence of the Ser120 side chain. We think that the correction of the folding defect by phosphorylation is due to the increase in the thermodynamic stability of phosphorylated M over that of I. We are currently searching for intragenic suppressors of the folding defect associated with the S120G mutation to confirm this hypothesis. Constantin Craescu for the preliminary NMR experiments.

. Effect of phosphorylation on the reactivation of the S120G mutant NDP kinase A. A)

The S120G mutant was phosphorylated with 100 µM ATP and the regeneration system described in the text. Phosphorylation was monitored with the lactate dehydrogenase reaction. Final protein concentrations were 0.54 mg/ml (wild-type), 0.49 mg/ml (S120G) and 0.44 mg/ml (H118N). B) Time-course for the reactivation of ATP-phosphorylated kinase (filled circles); ATP-phosphorylated enzyme, dephosphorylated with 100 µM ADP (empty circles); phosphoramidate-phosphorylated enzyme (filled squares); phosphoramidate-phosphorylated enzyme, dephosphorylated with 100 µM ADP (empty circles); and nonphosphorylated enzyme (triangles). Activity is expressed as % native NDP enzyme.

  . The reaction was pushed to completion by coupling with the pyruvate kinase reaction. The progress of phosphorylation was continuously monitored with the lactate dehydrogenase reaction. The phosphorylated protein was denatured by a 20 min incubation in the presence of solid GuHCl at a final concentration of 6 M. The denaturing reagent and ATP were eliminated rapidly on a HiTrap desalting column equilibrated with 100 mM Tris-HCl (pH 8.0). The final protein concentration was 20 µg/ml. The absence of ATP in the final preparation was ascertained by UV spectroscopy.

Fig 1 Biochemical

 1 Fig 1

  Second, in vivo, ATP concentration is high in all cells, with an [ATP]/[ADP] ratio between 5 and 10. The folding yield, in vivo, may depend on the actual [ATP]/[ADP] ratio. The folding intermediate may persist in cells that have a low [ATP]/[ADP] ratio. The intermediate may be trapped by chaperone proteins and have deleterious cellular effects.

Fig 3 The

 3 Fig 3

  S120G mutation decreases the equilibrium constant of the I <=> M reaction. How may phosphorylation assist subunit folding and assembly? The simplest hypothesis is that MgATP binds to the folded monomers and that protein autophosphorylation shifts the equilibrium towards M according to the mass action law. Subunit assembly into hexamers makes the entire reaction irreversible. ADP cancels, in part, the ATP effect by dephosphorylating M. The K-pn point mutation, P97S, in Drosophila NDP kinase does not affect folding, but does affect subunit association. The effect of the mutation cannot be corrected by phosphorylation in vitro (I. Lascu, unpublished results).

Fig 6 Finally

 6 Fig 6

Figure 2 .

 2 Figure 2. Effect of nucleotides on the reactivation of wild-type and S120G mutant NDP kinase A. A) Kinetics of reactivation. Urea-unfolded wild-type (squares) and S120G mutant (circles) NDP kinase A was diluted 20-fold in buffer in the absence (open symbols) or presence of 0.1 mM MgATP (filled symbols). The effects of 1 mM ADP (triangles) and of 1 mM AMP-PNP (diamonds) on the S120G mutant is also shown. The protein was used at a concentration of 5 µg/ml. B) Extent of S120G mutant reactivation after 2 hr in the presence of ATP (circles), ATP + 100 µM ADP (squares), ATP + 100 µM AMP-PNP (triangles) with 5 mM MgCl 2 (filled symbols) and 1 mM EDTA (open symbols). The reaction was followed in the presence of the indicated concentration. C) Reactivation of urea-unfolded S120G mutant diluted in buffer containing MgCl 2 . ATP, at a concentration of 100 µM, was present at time zero (filled circles) or was added after 30 min (empty circles). Triangles represent reactivation in the absence of ATP.

Figure 3 .

 3 Figure 3. Effect of MgATP on the renaturation/assembly of wild-type and mutant NDP kinase A, studied by size-exclusion chromatography. The urea-unfolded proteins (50 µl of 70 µg/ml solutions) were injected in a Superdex 75 column. Shown are the wild-type NDP kinase A (panel A), S120G mutant (panel B), H118N mutant (panel C) and the H118N/S120G double mutant (panel D). ATP was either absent (open symbols) or present at 100 µM The native enzymes were injected for control experiments (continuous line, no symbol). E) The elution profile of the phosphorylated enzyme, in the absence (filled circles) and presence (empty circles) of 100 µM ADP in the elution buffer. The ordinate axis represents the intrinsic protein fluorescence intensity, in arbitrary units.

Figure 4 .

 4 Figure 4. Effect of MgATP on urea-denatured NDP kinase A folding intermediate formation after dilution with buffer.Circles indicate the wild-type protein; squares, the S120G mutant; diamonds, the H118N mutant; and triangles, the H118N/S120G double mutant. The protein concentration was 10 µg/ml and the residual urea concentration was 0.4 M. MgATP was absent (filled circles) or present at 100 µM (open circles). a. u., arbitrary units.

Figure 5 .

 5 Figure 5. Effect of MgATP on folding intermediate accumulation during the renaturation of NDP kinase A. Urea-unfolded protein was diluted into buffer and incubated for 16 h in the indicated concentration of urea in the absence (filled symbols) or presence (empty symbols) of 100 µM MgATP. BisANS was added at a final concentration of 1 µM. A) Circles indicate wild-type NDP kinase and squares indicate S120G mutant NDP kinase. B) Diamonds indicate H118N mutant NDP kinase and triangles indicate H118N/S120G double mutant NDP kinase.

Figure 6 .

 6 Figure 6. Structure of the Drosophila NDP kinase phosphorylated intermediate. The PDB file 1NSQ coordinates were used to draw the figure. A) View of the hexamer along the 3-fold symmetry axis (only the top subunits are shown). B) Details of the phosphorylated active-site histidine environment.

Biochemical Journal Immediate Publication. Published on 8 Dec 2006 as manuscript BJ20061141

Biochemical Journal Immediate Publication. Published on 8 Dec 2006 as manuscript BJ20061141 Copyright 2006 Biochemical Society reactivation

  yield was noted when Mg 2+ ions were absent (with 1 mM EDTA in solution).These different effects suggest that it is protein phosphorylation and not nucleotide binding that corrects the folding defect. We investigated the effect of ADP on NDP kinase reactivation to confirm this hypothesis. The effect of ADP should be very different depending on whether NDP kinase reactivation occurs through enzyme phosphorylation or nucleotide binding. If the activation mechanism involves protein phosphorylation, ADP should have no effect by itself, but it should drive the equilibrium of reaction 1a to the left, dramatically decreasing the effect of MgATP. If reactivation is caused by nucleotide binding, ADP might have a weak activating effect as it binds to the enzyme. ADP should be a weak competitor against ATP because of their different affinities for NDP kinase; ATP binds NDP kinase with a much higher affinity (K d of 0.25 µM) than ADP (K d of 25 µM)

5 mM Mg 2+ at pH 8.0, and are referred to below as MgATP and MgADP, respectively. A low Fig 2

Biochemical Journal Immediate Publication. Published on 8 Dec 2006 as manuscript BJ20061141 Copyright 2006 Biochemical Society or

  with buffer containing 100 µM MgATP. The native protein served as a control. We identified the folding intermediate by mixing the column effluent with BisANS and measuring its fluorescence in a flow cuvette. BisANS, like ANS, binds specifically to folding intermediates and does not bind native or unfolded proteins[START_REF] Shi | Protein conformational changes induced by 1,1'bis(4-anilino-5-naphthalenesulfonic acid): preferential binding to the molten globule of DnaK[END_REF]. The binding of BisANS distinguished the folding intermediate from other forms that have a similar Stokes radius, for example the dimers. However,

	quantitative estimation was not possible because the increase in the hexamer peak was not
	accompanied by a proportional decrease in BisANS binding.
	The native wild-type NDP kinase A eluted as a hexamer at 11.0 ml (Fig. 3A). When unfolded
	protein was injected, most of the sample recovered the hexameric structure. The shoulder observed
	at 12.2 ml corresponds to a folding intermediate which binds BisANS. We described previously the
	peculiar folding/association pattern of human NDP kinase A [22, 32, 33]. Typical hexameric NDP

kinases, including the Dictyostelium cytosolic and Drosophila enzymes, unfold without the accumulation of dissociated species. In the refolding experiment, they fold as native monomers, which subsequently associate into hexamers. However, during the folding of the human NDP kinase A it is an equilibrium folding intermediate that accumulates, not folded monomers. The native S120G mutant and the native wild-type enzyme eluted at with the same profile, indicating that the S120G mutant is a hexamer in solution, as in the crystal. We did not detect dissociated or BisANSbinding forms. This is a diagnostic test for the quality of the protein preparation. Following injection of the unfolded S120G mutant protein in the absence of MgATP we only detected the folding intermediate, characterized by BisANS binding (not shown). MgATP promotes correct folding and association (Fig.

3B

). Control experiments showed that ATP in the absence of Mg 2+ , and ADP in the presence or absence of Mg 2+ have no effect.

Biochemical Journal Immediate Publication. Published on 8 Dec 2006 as manuscript BJ20061141

  [START_REF] Andersson | Cofactor-induced refolding: refolding of molten globule carbonic anhydrase induced by Zn(II) and Co(II)[END_REF] demonstrated that Zn 2+ and Co 2+ induced refolding of carbonic anhydrase by their interactions with the molten globule state[START_REF] Andersson | Cofactor-induced refolding: refolding of molten globule carbonic anhydrase induced by Zn(II) and Co(II)[END_REF]. It has been recently shown that metal ions interact with the unfolded form of the protein and promote folding into the native form rather than the folding

Biochemical Journal Immediate Publication. Published on 8 Dec 2006 as manuscript BJ20061141

Biochemical Journal Immediate Publication. Published on 8 Dec 2006 as manuscript BJ20061141 Copyright 2006 Biochemical Society


Guy Martin

Bernard Ferrier

Agnès Conjard

Mireille Martin

Rémi Nazaret

Michelle Boghossian

Fadi Saadé

Claire Mancuso

Daniel Durozard

Pr G Baverel
email: baverel@sante.univ-lyon1.fr

Glutamine gluconeogenesis in the small intestine of 72h-fasted adult rats is undetectable

Keywords: glutamine metabolism, glucose synthesis, ketone bodies Biochemical

Recent reports have indicated that 48-72h of fasting, type 1 diabetes and high-protein feeding induce gluconeogenesis in the small intestine of adult rats in vivo. Since this would (i) represent a dramatic revision of the prevailing view that only the liver and the kidneys are gluconeogenic, and (ii) have major consequences in the metabolism, nutrition and diabetes fields, we have thoroughly reexamined this question in the situation reported to induce the highest rate of gluconeogenesis. For this, metabolically viable small intestinal segments from 72h-fasted adult rats were incubated with [3-13 C]glutamine as substrate. After incubation, substrate utilization and product accumulation were measured by enzymatic and NMR spectroscopic methods. Although the segments utilized 13 Cglutamine at high rates and accumulated 13 C-labelled products linearly for 30 min in vitro, no substantial glucose synthesis could be detected. This was not due to the re-utilization of 13 C-glucose initially synthesized from 13 C-glutamine. Arteriovenous metabolite concentration difference measurements across the portal vein-drained viscera of 72h-fasted Wistar and Sprague-Dawley rats clearly indicated that glutamine, the main if not the only gluconeogenic precursor taken up, could not give rise to detectable glucose production in vivo. Therefore, we challenge the view that the small intestine of the adult rat is a gluconeogenic organ.

Introduction

The contribution of the liver to glucose production in normal fasted humans and animals is well established; under this situation, the liver produces glucose as a result of both gluconeogenesis mainly from lactate, alanine, glutamine and glycerol, and glycogenolysis when glycogen stores are available [START_REF] Nordlie | Fine tuning of blood glucose concentrations[END_REF][START_REF] Radziuk | Hepatic glucose uptake, gluconeogenesis and the regulation of glycogen synthesis[END_REF]. Various studies performed in humans and, to a lesser extent, in rats have suggested that, besides the liver, the kidneys may contribute to systemic gluconeogenesis; indeed, it has been reported that renal gluconeogenesis may represent 5 to 45% of systemic gluconeogenesis in postabsorptive or fasted humans (see 3 for a recent review).

Combining isotopic techniques with or without arteriovenous concentration difference measurements or organ vessel ligature, Mithieux and co-workers recently published a series of articles assessing the contribution of the small intestine of adult rats to systemic endogenous glucose production under various experimental conditions [START_REF] Croset | Rat small intestine is an insulin-sensitive gluconeogenic organ[END_REF][START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF][START_REF] Mithieux | Portal sensing of intestinal gluconeogenesis is a mechanistic link in the diminution of food intake induced by diet protein[END_REF][START_REF] Mithieux | Contribution of intestine and kidney to glucose fluxes in different nutritional states in rat[END_REF]. In agreement with the findings of Watford et al. [START_REF] Watford | Isolation and metabolic characteristics of rat and chicken enterocytes[END_REF] and of Baverel and Lund [START_REF] Baverel | A role for bicarbonate in the regulation of mammalian glutamine metabolism[END_REF] with isolated enterocytes from fed rats, they did not observe any intestinal gluconeogenesis in vivo in control rats fasted for 5-6 or 24 hours [START_REF] Croset | Rat small intestine is an insulin-sensitive gluconeogenic organ[END_REF][START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF] although they more recently found the reverse in 24h-fasted animals [START_REF] Mithieux | Portal sensing of intestinal gluconeogenesis is a mechanistic link in the diminution of food intake induced by diet protein[END_REF][START_REF] Mithieux | Contribution of intestine and kidney to glucose fluxes in different nutritional states in rat[END_REF]. In addition, no intestinal glucose synthesis was observed in post-aborptive or 24h-fasted rats in vivo, especially from 14 C-glutamine [START_REF] Croset | Rat small intestine is an insulin-sensitive gluconeogenic organ[END_REF][START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF]. By contrast, they concluded from their data that the small intestine of the rat accounted for 21%, 35%, 19% and 19 % of systemic gluconeogenesis after 48 h or 72h of fasting, after 3 days of type 1 diabetes and 2-15 days of a high-protein diet, respectively [START_REF] Croset | Rat small intestine is an insulin-sensitive gluconeogenic organ[END_REF][START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF][START_REF] Mithieux | Portal sensing of intestinal gluconeogenesis is a mechanistic link in the diminution of food intake induced by diet protein[END_REF][START_REF] Mithieux | Contribution of intestine and kidney to glucose fluxes in different nutritional states in rat[END_REF]. These authors also found that, in the rat small intestine, an increase in the expression and activity of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, two key-gluconeogenic enzymes, was consistent with the in vivo induction of gluconeogenesis [START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF][START_REF] Rajas | The glucose-6 phosphatase gene is expressed in human and rat small intestine: regulation of expression in fasted and diabetic rats[END_REF][START_REF] Rajas | Induction of PEPCK Biochemical Journal Immediate Publication[END_REF]. Furthermore, they claimed that the satiety induced by a highprotein diet was linked to the increase in intestinal gluconeogenesis caused by such a diet [START_REF] Mithieux | Portal sensing of intestinal gluconeogenesis is a mechanistic link in the diminution of food intake induced by diet protein[END_REF]. Thus, in the light of the latter studies, intestinal gluconeogenesis would become a central process controlling not only glucose homeostasis but also the whole energy metabolism of the body. The observations and conclusions of Mithieux and co-workers are intriguing because, unlike the liver and the kidney, the small intestine of the rat has until recently never been considered to be a site of intense gluconeogenic capacity. To our knowledge, only Windmueller and Spaeth previously observed in vivo that the small intestine of the adult rat has a very low capacity to synthesize radioactive glucose from radioactive aspartate, glutamine and lactate [START_REF] Windmueller | Metabolism of absorbed aspartate, asparagine, and arginine by rat small intestine in vivo[END_REF][START_REF] Windmueller | Identification of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestine[END_REF]. In vitro preparations of the small intestine from suckling but not weaned rats have also been reported to synthesize some glucose from lactate [START_REF] Hahn | Gluconeogenesis from lactate in the small intestinal mucosa of suckling rats[END_REF].

Given (i) the potential importance of the small intestine as a gluconeogenic organ and as the source of major glucose homeostasis disturbances, (ii) the absence of glucose synthesis from glutamine in isolated enterocytes from fed rats shown by Watford et al. and Baverel and Lund [8,[START_REF] Baverel | A role for bicarbonate in the regulation of mammalian glutamine metabolism[END_REF], (iii) that the conclusions drawn by Mithieux and co-workers should be taken with caution, as pointed out by the authors themselves because of inaccuracies inherent to the numerous experimental steps needed to determine the gluconeogenic rates they reported [START_REF] Mithieux | A novel role for glucose 6-phosphatase in the small intestine in the control of glucose homeostasis[END_REF] and (iv) the recent review by Watford casting serious doubt on the existence of intestinal gluconeogenesis [START_REF] Watford | Is the small intestine a gluconeogenic organ?[END_REF], we have decided to thoroughly reexamine this question both in vitro and in vivo. For this, we have isolated metabolically viable segments of the small intestine obtained from rats subjected to a 72-h fast, an experimental condition reported so far to be the most potent inducer of intestinal gluconeogenesis [START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF]. These segments, made of the entire intestinal wall to mimick in vitro the structure of the small intestine in vivo, were incubated with 13 C-labelled glutamine. Taking advantage of the 13 C NMR spectroscopy and enzymatic approaches that enabled us to unravel the complexity of the renal metabolism of various substrates [START_REF] Chauvin | The rabbit kidney tubule utilizes glucose for glutamine synthesis. A 13C NMR study[END_REF][START_REF] Chauvin | The rabbit kidney tubule simultaneously degrades and synthesizes glutamate. A 13C NMR study[END_REF][START_REF] Dugelay | Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study[END_REF][START_REF] Conjard | The anaplerotic substrate alanine stimulates acetate incorporation into glutamate and glutamine in rabbit kidney tubules. A 13C-NMR study[END_REF][START_REF] Vercoutère | Complexity of glutamine metabolism in kidney tubules from fed and fasted rats[END_REF][START_REF] Vittorelli | Characteristics of Biochemical Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148 Copyright 2006 Biochemical Society glutamine metabolism in human precision-cut kidney slices: a 13C-NMR study[END_REF], we measured glutamine utilization and product accumulation. Despite high rates of glutamine metabolism, substantial glucose synthesis could not be detected. Furthermore, arteriovenous concentration difference measurements were performed across the intestine not only in 72h-fasted Wistar but also in 72h-fasted Sprague Dawley rats because the latter rat strain was used by Mithieux and co-workers [START_REF] Croset | Rat small intestine is an insulin-sensitive gluconeogenic organ[END_REF][START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF][START_REF] Mithieux | Portal sensing of intestinal gluconeogenesis is a mechanistic link in the diminution of food intake induced by diet protein[END_REF][START_REF] Mithieux | Contribution of intestine and kidney to glucose fluxes in different nutritional states in rat[END_REF]. The results obtained revealed that the small percentage of glutamine taken up by the intact intestine cannot explain the large glucose production reported by Mithieux and co-workers in their rats under the same experimental conditions. A preliminary account of part of the present results has been published as an abstract [START_REF] Baverel | In vitro, la glutamine est un précurseur de glucose dans les cellules hépatiques et rénales mais pas dans les cellules intestinales de rats nourris, à jeun et diabétiques[END_REF].

Methods

Reagents

Glucosamine and glutaminase (grade V) were from Sigma (St Quentin-Fallavier, France). Other enzymes and coenzymes were purchased from Boehringer Mannheim (Meylan, France). L-[3- 13 C]glutamine and D-[2-13 C]glucose were obtained from Eurisotop (Saclay, France) and had a 99% isotopic abundance.

Rats

All experiments were approved by the Institutional Animal Care and Use Committee of the Lyon 1 University. Male Wistar or Sprague-Dawley rats weighing between 250 and 300 g were obtained from Charles River (Saint-Germain-sur-l'Arbresle, France). They were acclimated to our animal house for one week, kept on a 12/12h light-dark cycle, and fed a standard diet (U.A.R., Villemoisson-sur-Orge, France). Then, they were fasted for 72 h before the experiments but had free access to water.

Preparation and incubation of intestinal segments from 72h-fasted Wistar rats

Each rat was anaesthetized with intraperitoneal Nembutal (50 mg.kg body weight -1 ). The abdomen was opened and the small intestine was exteriorized, quickly excised and stripped away from the mesentery. The lumen of the small intestine was flushed out with twice 60 ml of ice-cold oxygenated Krebs-Henseleit medium. All the subsequent steps of preparation were performed in cold oxygenated Krebs-Henseleit buffer. Then, rings of 2-4 mm width were cut with scissors from the entire length of the small intestine. The rings were pooled and randomized in a flask and then separated from the buffer on a sieve, gathered on a cold glass surface and divided in 5 equivalent batches of rings that were immediately incubated. Two batches of rings were incubated per incubation time ( experiments. The flasks were incubated at 37°C in a shaking water bath. Incubation was terminated by addition of perchloric acid [final concentration 5% (v/v)] and rapid homogeneization of tissue plus medium. A batch of rings was taken to determine the initial amounts of added and endogenous substrates by adding perchloric acid before the rings. After centrifugation (3,000 g for 5 min), the denaturated rings were used for measurement of their protein content; the supernatant was neutralized with a mixture of 20% (w/v) KOH and 1% (v/v) H 3 PO 4 and used for metabolite determination and NMR spectroscopy.

Measurement of arteriovenous concentration differences

Anaesthetized, 72h-fasted Wistar and Sprague-Dawley rats (250-300g), were placed on a heated table to maintain their body temperature at 37-38°C. Then, a catheter was inserted into the carotid artery. After laparotomy, the gut was gently moved on the left side and covered with gauze moistened with warm saline. Hepatic portal blood was collected in the following way: a needle (gauge 23) was bent, mounted on a pipette holder, connected to a syringe by a length of polyethylene tubing and introduced into the portal vein by puncture. For blood withdrawing, the position of the needle tip inside the hepatic portal vein was continuously controlled thanks to a binocular lens with a magnification of 8-fold. Blood samples (1.5 ml each) were aspirated slowly (over 60 seconds) and simultaneously from the carotid artery and the hepatic portal vein. This procedure was never accompanied by any bleeding. Immediately after blood collection, 1.3 ml of blood samples was deproteinized with 0.5 ml of 20% (v/v) chilled perchloric acid. After centrifugation, the supernatants were neutralized with a mixture of KOH and phosphoric acid as described above and used for metabolite analysis. The intestinal tissue (rings) ATP concentration was quantified by using the method of Lamprecht and Trautschold [START_REF] Lamprecht | Adenosine-5'-triphosphate. Determination with hexokinase and glucose-6-phosphate dehydrogenase[END_REF]. Pellets were solubilized in 0.5 M NaOH for protein determination and total protein was determined as described previously [START_REF] Conjard | Effect of starvation on glutamine ammoniagenesis and gluconeogenesis in isolated mouse kidney tubules[END_REF].

Analytical methods

-ATP and protein content

-Metabolite assays Glutamine, glutamate, alanine, lactate, pyruvate, aspartate, glucose, ammonia, glycerol, D-βhydroxybutyrate, acetoacetate and glycogen were determined according to Passonneau and Lowry [START_REF] Passonneau | Enzymatic Analysis: A Practical Guide[END_REF].

- 13 C NMR techniques Data were recorded as indicated previously [START_REF] Chauvin | The rabbit kidney tubule utilizes glucose for glutamine synthesis. A 13C NMR study[END_REF][START_REF] Chauvin | The rabbit kidney tubule simultaneously degrades and synthesizes glutamate. A 13C NMR study[END_REF][START_REF] Vittorelli | Characteristics of Biochemical Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148 Copyright 2006 Biochemical Society glutamine metabolism in human precision-cut kidney slices: a 13C-NMR study[END_REF] at 125.75 MHz on a Bruker AM-500 WB spectrometer using a 5 mm broadband probe thermostated at 8 ± 0.5°C, except that the number of scans was 420. Chemical shifts were expressed as parts/million (ppm) relative to tetramethylsilane.

Assignments were made by comparing the chemical shifts obtained with those given in the literature [START_REF] Howarth | Carbon-13-NMR of peptides and proteins[END_REF]29]. Given that glutamine gluconeogenesis from [3-13 C]glutamine leads to the equal labelling of the C-1, C-2, C-5 and C-6 of glucose [START_REF] Vercoutère | Complexity of glutamine metabolism in kidney tubules from fed and fasted rats[END_REF], and taking a signal/noise ratio equal to 1.5 for the heights of the corresponding β resonances of glucose carbons, our calculations indicate that, under our experimental conditions, approximately 0.7 µmol of the C-3 of [3-13 C]glutamine had to be converted into glucose to enable us to detect glutamine gluconeogenesis.

Calculations and statistical analysis

Net substrate utilization and product formation were calculated as the difference between the total flask contents (tissue plus medium) at the start (zero-time flasks) and after the period of incubation. The net metabolic rates, reported as means ± S.E.M., are expressed in µmol of substance removed or produced per unit time (20 or 30 min) per g of intestinal tissue protein. With [3-13 C]glutamine as substrate, the transfer of the C-3 of glutamine to a given position in a given metabolite was calculated by using the formula described previously [START_REF] Chauvin | The rabbit kidney tubule utilizes glucose for glutamine synthesis. A 13C NMR study[END_REF][START_REF] Chauvin | The rabbit kidney tubule simultaneously degrades and synthesizes glutamate. A 13C NMR study[END_REF][START_REF] Vittorelli | Characteristics of Biochemical Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148 Copyright 2006 Biochemical Society glutamine metabolism in human precision-cut kidney slices: a 13C-NMR study[END_REF].

The blood metabolite concentrations found in the carotid artery and the hepatic portal vein were compared using the paired Student's t test . P<0.05 was considered to be statistically significant.

Results

Glutamine metabolism in intestinal segments from 72h-fasted rats

Since the viability of isolated rat enterocytes is limited with time [START_REF] Watford | Isolation and metabolic characteristics of rat and chicken enterocytes[END_REF], our segments were incubated for 20 and 30 min, but, for accuracy purposes, these short incubation times were compensated by the incubation of large amounts of intestinal tissue in a large volume (20 ml) of incubation medium. At zero-time, the ATP content of intestinal segments was 2.99 ± 0.71 µmol.g protein -1 (n = 3). In intestinal segments incubated with glutamine, the ATP content was 2.90 ± 0.49 and 2.90 ± 0.39 µmol.g protein -1 after 20 and 30 min of incubation, respectively (n = 3).

Table 1 shows the time-course of glutamine utilization and product formation measured by enzymatic methods when the segments were incubated with 5 mM [3-13 C]glutamine as substrate.

Glutamine utilization occurred at high rates and in a linear fashion with time. The rate of glutamate accumulation was approximately constant whereas that of alanine, lactate and ammonia tended to increase with time. Small amounts of aspartate, urea and pyruvate accumulated and negligible amounts of glucose were synthesized. In the absence of glutamine, substantial amounts of alanine, lactate and ammonia, and very small amounts of glucose accumulated from endogenous substrates (Table 1). From the spectra obtained after 20 and 30 min of incubation of segments from 72h-fasted rats, we calculated the amounts of [3-13 C]glutamine removed and of labelled products accumulated after correction for the 13 C natural abundance. As shown in Table 2, the removal of the labelled glutamine was also approximately linear with time and this removal was not statistically different with that measured enzymatically (see Table 1). This means that, in agreement with the very low activity of glutamine synthetase in the small intestine [START_REF] James | Glutamine metabolism in the gastrointestinal tract of the rat assessed by the relative activities of glutaminase (EC 3.5.1.2) and glutamine synthetase (EC 6.3.1.2)[END_REF], there was no substantial glutamine synthesis in our experiments. The labelling of glutamate, alanine and lactate was lower than the amounts of these products found to accumulate (see Table 1); this was especially true for alanine and to a lesser extent for glutamate and lactate. The fact that the labelling of aspartate was higher than the net aspartate accumulation presented in Table 1 simply reflects the fact that the unlabelled aspartate brought by the segments at zero-time was metabolized and replaced by newly synthesized labelled aspartate.

Table 2 also shows that the labelled C-2 of aspartate, alanine and lactate was virtually equal to the labelled C-3 of these compounds; this is in agreement with the view that the C-3 of glutamine converted into the C-3 of glutamate by glutaminase, and then into the C-3 of 2-oxoglutarate by alanine, aspartate and ornithine aminotransferases, was further metabolized in the tricarboxylic acid cycle via succinate and fumarate, two symmetrical molecules. The passage through these symmetrical molecules is also consistent with the labelling of the C-2 of glutamate which involves one complete turn of the tricarboxylic acid cycle. The labelling of the C-1 of lactate and alanine is also consistent with the recycling of the C-3 of glutamine through the tricarboxylic acid cycle to yield the C-1 of malate and then the C-1 of pyruvate by malic enzyme, or the C-1 of oxaloacetate and then the C-1 of pyruvate by oxaloacetate decarboxylase or phosphoenolpyruvate carboxykinase plus pyruvate kinase [START_REF] Watford | Isolation and metabolic characteristics of rat and chicken enterocytes[END_REF]. In agreement with the results of Windmueller and Spaeth who used 14 Cglutamine as substrate [START_REF] Windmueller | Identification of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestine[END_REF][START_REF] Windmueller | Uptake and metabolism of plasma glutamine by the small intestine[END_REF]32], some 13 C-labelled ornithine, citrulline and proline were also formed from 13 C-labelled glutamine. It is noteworthy that the sum of the non-volatile labelled Copyright 2006 Biochemical Society products of 13 C-glutamine metabolism increased linearly with time. Neglecting the possibility that a small fraction of the 13 C-glutamine removed was incorporated into tissue materials [START_REF] Windmueller | Identification of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestine[END_REF][START_REF] Windmueller | Uptake and metabolism of plasma glutamine by the small intestine[END_REF]32], we calculated the 13 CO 2 produced from [3-13 C]glutamine to estimate the complete oxidation of glutamine carbon skeletons as explained in a previous publication [START_REF] Vercoutère | Complexity of glutamine metabolism in kidney tubules from fed and fasted rats[END_REF]. Both after 20 and 30 min of incubation, approximately half of the C-3 of glutamine used was converted into CO 2 (59.4 ±8.9 and 81.2 ± 11.2 µmol.g protein -1 . incubation time -1 , respectively). The corresponding proportions of glutamine carbon converted into CO 2 are close to those found in the rat small intestine in vivo [START_REF] Windmueller | Identification of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestine[END_REF][START_REF] Windmueller | Uptake and metabolism of plasma glutamine by the small intestine[END_REF]32] indicating that all steps of the tricarboxylic acid cycle were well functional in our segments in vitro.

Evidence that gluconeogenesis from glutamine in small intestinal segments from 72h-fasted

Wistar rats was not masked by concomitant glucose re-utilization

Given the high capacity of the small intestine to metabolize glucose in vitro [START_REF] Watford | Isolation and metabolic characteristics of rat and chicken enterocytes[END_REF][START_REF] Dickens | Metabolism of normal and tumour tissue[END_REF]34], we used two experimental approaches to test the possibility that the 13 C-glucose synthesized from part of the [3- 13 C]glutamine metabolized by our intestinal fragments was re-utilized via the glycolytic pathway. As done in a recent study performed with precision-cut human kidney slices [START_REF] Vittorelli | Characteristics of Biochemical Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148 Copyright 2006 Biochemical Society glutamine metabolism in human precision-cut kidney slices: a 13C-NMR study[END_REF], we hypothesized that the [3-13 C]glutamine not accounted for by the non-volatile 13 C-products found to accumulate might have been initially converted into 13 C-glucose and then re-utilized and oxidized into 13 CO 2 by our intestinal segments. It can be calculated that, if all this synthesized 13 C-glucose had accumulated in the incubation medium, its concentration would have been approximately 1 mM.

-Effect of glucosamine on the metabolism of 5 mM [3-13 C]glutamine

First, we tested the efficacy of glucosamine, an inhibitor of hexokinase [START_REF] Guglielminetti | Characterization of isoforms of hexose kinases in rice embryo[END_REF], on glucose utilization by small intestinal segments from 72h-fasted Wistar rats. With 1 mM D-[2- 13 C]glucose as substrate, addition of 40 mM glucosamine induced a mean 34% inhibition of the removal of [2- 13 Copyright 2006 Biochemical Society µmol.g.protein -1 .30 min -1 in the absence and the presence of glucosamine, respectively (n = 3; P < 0.05). Fig. 2 shows a 13 C NMR spectrum obtained in the presence of 40 mM glucosamine in one representative experiment out of two performed in duplicate in which small intestinal segments from 72h-fasted Wistar rats were incubated for 30 min with 5 mM [3- 13 C]glutamine in the absence and the presence of 40 mM glucosamine. This spectrum, which shows the resonances corresponding to the C-3 of glutamine and those of the 13 C-labelled products of 13 C-glutamine (as in Fig. 1) and the resonances of the glucosamine carbons, clearly demonstrates that there were no glucose carbon resonances. In this respect, it should be pointed out that the resonances of the C-1 of glucose, which are well separated from those of the C-1 of glucosamine, were absent.

-Effect of 5 mM glucose on the metabolism of 5 mM [3-13 C]glutamine in small intestinal segments from 72h-fasted Wistar rats

Then, with the hope that large amounts of glucose added at zero-time would (i) dilute the 13 Clabelled glucose possibly synthesized from [3-13 C]glutamine and (ii) prevent its complete reutilization, four additional experiments were performed with small intestinal segments (265.8 ± 8.0 mg protein.flask-1) from 72h-fasted Wistar rats incubated for 30 min with 5 mM [3-13 C]glutamine in the absence and the presence of 5 mM unlabelled D-glucose. Under the latter conditions, glutamine utilization was 136.5 ± 8.9 and 142.4 ± 10.1 µmol.g.protein -1 .30 min -1 , respectively. In the presence of 5 mM glucose, glucose utilization was 148.8 ± 15 µmol.g.protein -1 .30 min -1 . Fig. 3 presents a 13 C NMR spectrum obtained in the presence of 5 mM [3-13 C]glutamine + 5 mM unlabelled D-glucose in one representative experiment. This spectrum, which shows the resonance of the C-3 of glutamine and those of the 13 C-labelled products of 13 C-glutamine metabolism (as in Fig. 1

Metabolite concentrations in arterial and portal vein blood of 72h-fasted Wistar and Sprague-

Dawley rats

Table 3 shows the mean concentrations of all the metabolite measured in the arterial and portal vein blood of 72h-fasted rats. In both strains, there was a net uptake of glucose; this markedly contrasts with the absence of net glucose uptake or release observed by Mithieux and co-workers in their 72hfasted Sprague-Dawley rats [START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF]. In agreement with the classical results obtained by numerous authors in fed rats or in rats fasted for up to 48 hours [START_REF] Windmueller | Identification of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestine[END_REF][START_REF] Windmueller | Uptake and metabolism of plasma glutamine by the small intestine[END_REF][START_REF] Ardawi | Glutamine and ketone-body metabolism in the gut of streptozotocindiabetic rats[END_REF][START_REF] Brosnan | Interorgan metabolism of amino acids in streptozotocin-diabetic ketoacidotic rat[END_REF][START_REF] Watford | The regulation of phosphate-activated glutaminase activity and glutamine metabolism in the streptozotocin-diabetic rat[END_REF][START_REF] Welbourne | Regulation of interorganal glutamine flow in metabolic acidosis[END_REF], we observed a 30% uptake of arterial glutamine by the intestine in our 72h-fasted Wistar rats whereas the corresponding value was only 18% in our 72h-fasted Sprague-Dawley rats.

A statistically significant release of glutamate occurred only in Sprague-Dawley rats and this release represented 60% of the glutamine taken up in these animals. A release of alanine, which was in the same order of magnitude in Wistar and Sprague-Dawley rats, was observed.

In agreement with the observation of Windmueller and Spaeth [START_REF] Windmueller | Identification of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestine[END_REF] in their overnight-fasted Osborne-Mendel rats when arterial lactate was higher than 1.2-1.6 mM, the intestine of our Wistar rats took up a large fraction (37%) of circulating lactate; by contrast, the intestine of our Sprague-Dawley rats neither took up nor released lactate in a statistically significant manner. A very small uptake of glycerol occurred only in Sprague-Dawley rats. D-β-hydroxybutyrate, whose arterial concentration was much higher in Wistar than in Sprague-Dawley rats was taken up only by the intestine of Wistar rats and part of this D-β-hydroxybutyrate was released as acetoacetate which was neither taken up nor released by the intestine of Sprague-Dawley rats.

Discussion

Although we used Wistar rats after 72h of fasting, an experimental condition reported in vivo to induce a large intestinal gluconeogenesis [START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF], the results of the present study unequivocally demonstrate that, when incubated in vitro, the small intestine of our animals did not synthesize glucose from glutamine, the main if not the exclusive gluconeogenic precursor after 48h [START_REF] Croset | Rat small intestine is an insulin-sensitive gluconeogenic organ[END_REF] and presumably 72h of fasting. In that respect, it resembles the isolated perfused and intact functioning small intestine of overnight-fasted rats [START_REF] Windmueller | Uptake and metabolism of plasma glutamine by the small intestine[END_REF]32] and the enterocytes isolated from fed rats in which no glutamine gluconeogenesis was reported [START_REF] Watford | Isolation and metabolic characteristics of rat and chicken enterocytes[END_REF][START_REF] Baverel | A role for bicarbonate in the regulation of mammalian glutamine metabolism[END_REF]. It should be mentioned here that, within the intestinal wall, the enterocytes are the only site of glutamine metabolism [START_REF] Windmueller | Uptake and metabolism of plasma glutamine by the small intestine[END_REF]. Moreover, our in vivo results do not corroborate those of Mithieux and co-workers who concluded that the small intestine of 72hfasted Sprague-Dawley rats is responsible for at least one-third (35%) of systemic gluconeogenesis [START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF].

Metabolic viability of our small intestinal fragments and absence of glucose synthesis

Several lines of evidence indicate that our intestinal segments, which consisted of the entire small intestinal wall, were metabolically viable. Taking a dry weight/fresh weight ratio equal to 0.23 [START_REF] Windmueller | Uptake and metabolism of plasma glutamine by the small intestine[END_REF] and assuming that 90% of the dry weight consisted of protein, it can be calculated that the small intestine preparation used by Windmueller and Spaeth [32] metabolized the luminally added glutamine (6 mM) at a rate of 27 µmol.g protein -1 .30min -1 . Taking the glutamine removed in our experiments after 30 min of incubation (Table 1), it can also be calculated that our small intestinal segments, whose a maximum of 40% consisted of enterocytes [START_REF] Kong | Starvation alters the activity and mRNA level of glutaminase and glutamine synthetase in the rat intestine[END_REF][START_REF] Mcmanus | Effect of fasting versus feeding on the rat small intestine. Morphological, biochemical, and functional differences[END_REF], metabolized 5 mM glutamine at a much higher rate (586 µmol.g protein -1 .30 min -1 ). The enterocytes isolated by Watford et al. [START_REF] Watford | Isolation and metabolic characteristics of rat and chicken enterocytes[END_REF] and by Baverel and Lund [9] metabolized 5 mM glutamine at a rate of 367 and 300 µmol.g protein - 1 .30min -1 , respectively. Thus, although our measurements and calculations reveal that the ATP level of our segments was only about one half that found in vivo [START_REF] Zager | Adenine nucleotide changes in kidney, liver, and small intestine during different forms of ischemic injury[END_REF], and although the enterocytes represented only about 40% of our intestinal segments, they were metabolically very active and 1 and2). It is also important to note that, like the glutamine taken up from the lumen and from the circulating blood in the in vivo preparations used by Windmueller and Spaeth [START_REF] Windmueller | Identification of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestine[END_REF][START_REF] Windmueller | Uptake and metabolism of plasma glutamine by the small intestine[END_REF], about half of our glutamine metabolized was completely oxidized (see the Results section). This means that the mitochondria of our intestinal segments functioned satisfactorily. That the entire tricarboxylic acid cycle was operating in a satisfactory manner is also indicated by the conversion of the C-3 of glutamine not only into the C-2 and C-3 of alanine and lactate but also into the C-2 of glutamate (Fig. 1 and Table 2). Thus, the absence of substantial glucose synthesis in these segments was not due to a limitation in the provision of ATP but rather to the intrinsic organization of the glutamine metabolic pathways of rat enterocytes. It should also be mentioned here that Windmueller and Spaeth have concluded from their experiments that the glutamines taken up from the lumen on the one hand and, on the other hand, from blood shared a common metabolic pool [32]. Thus, our results obtained in vitro with luminally added glutamine can be considered relevant to the in vivo situation.

The very small amounts of glucose synthesized , which were not higher in the presence than in the absence of glutamine, presumably originated from the degradation of the very small amount of glycogen present in the intestinal segments of 72h-fasted rats (13 µmol glucosyl equivalents.entire small intestine -1 ).

Similarly, the absence of glutamine gluconeogenesis in our intestinal segments was not due to the reutilization of 13 C-glucose initially synthesized from 3-13 C-glutamine as strongly suggested not only by the use of glucosamine, an inhibitor of hexokinase [START_REF] Guglielminetti | Characterization of isoforms of hexose kinases in rice embryo[END_REF], but also by the incubation of 13 Cglutamine with a physiological concentration (5 mM) of glucose (see Fig. 2 and3). Under the latter experimental condition (see the Results section), a strong line of evidence that no synthesis of 13 Cglucose from [3-13 C]glutamine occurred is that the resonance intensities and areas (α + β anomers) of the six glucose carbons were equal (see Fig. 3 and the Results section); indeed, as recently shown in rat renal proximal tubules, gluconeogenesis from [3-13 C]glutamine leads to glucose carbons labelled with carbon 13 to different degrees [START_REF] Vercoutère | Complexity of glutamine metabolism in kidney tubules from fed and fasted rats[END_REF]. It should also be mentioned here that enterocytes Copyright 2006 Biochemical Society prepared from the small intestine adapted to a high-protein diet did not produce glucose from glutamine [START_REF] Azzout-Marniche | Glucoregulation in rat adapted to a high protein diet[END_REF].

Arteriovenous concentration difference measurements

To our knowledge, our study is the first to report arteriovenous concentration difference measurements across portal-drained viscera of 72h-fasted rats. It should be pointed out that, unlike Mithieux and co-workers in their in vivo experiments [START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF], we did not ligature the inferior mesenteric artery in order to avoid surgical stress. But this is unimportant with respect to glutamine metabolism because it occurs primarily in the small intestine; indeed, 87 % of glutaminase is located in the small intestine [START_REF] Pinkus | Phosphate-dependent glutaminase of small intestine: localization and role in intestinal glutamine metabolism[END_REF] and therefore, as noted by Windmueller and Spaeth [32], most of the glutamine uptake in our rats occurred in the small intestine.

After 72h of fasting, our observation of a net intestinal uptake of glucose in both Wistar and Sprague-Dawley rats (Table 3) markedly contrasts with the findings of Mithieux and co-workers who reported no net production or uptake of glucose by the small intestine of both 48h-and 72h-fasted Sprague-Dawley rats [START_REF] Croset | Rat small intestine is an insulin-sensitive gluconeogenic organ[END_REF][START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF]. However, this is in agreement with the observation of Burrin et al. who also found no evidence of gluconeogenesis in the small intestine of 36h-fasted pigs [START_REF] Burrin | Gastrointestinal gluconeogenesis is absent in the 36-hour fasted piglet[END_REF]. Since a very small, virtually negligible, glycerol uptake was observed only in Sprague-Dawley rats (Table 3), this means that the small intestine in vivo cannot synthesize substantial amounts of glucose from this substrate.

-Wistar rats: Considering that two glutamine molecules are needed to synthesize one glucose molecule, and neglecting the fact that about half the circulating glutamine taken up by the intestine was oxidized to CO 2 , one may calculate from our data that the arterial glucose entering the intestine would have been diluted by only 1.2 % (0.174:2:7.51) by the glutamine-derived glucose; the corresponding value would have been only 0.6% if one considers that half the glutamine taken up was oxidized to CO 2 . Unfortunately, Mithieux and co-workers did not report the intestinal substrate uptake in their experiments [START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF]. In agreement with observations of Windmueller and Spaeth when the Copyright 2006 Biochemical Society blood lactate was elevated [START_REF] Windmueller | Identification of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestine[END_REF], substantial amounts of lactate were taken up by the intestine of our 72h-fasted Wistar rats, but this potential gluconeogenic substrate could not have been converted into glucose because of the extremely low pyruvate carboxylase activity measured in the small intestine of the adult rat [START_REF] Anderson | Pyruvate carboxylase and phosphoenolpyruvate carboxykinase in rat intestinal mucosa[END_REF][START_REF] Hahn | Pyruvate carboxylase, phosphate-dependent glutaminase and glutamate dehydrogenase in the developing rat small intestinal mucosa[END_REF]. In addition, Mithieux and co-workers did not observe any conversion of 14 Clactate into 14 C-glucose in the small intestine of 48h-fasted rats in vivo [START_REF] Croset | Rat small intestine is an insulin-sensitive gluconeogenic organ[END_REF]. Furthermore, our preliminary results in vitro indicate that 5 mM 13 C-lactate is not converted into 13 C-glucose in intestinal segments prepared from 72h-fasted Wistar rats (results not shown).

-Sprague-Dawley rats: It should be pointed out that, in our 72h-fasted Sprague-Dawley rats, glutamine was the sole potential gluconeogenic precursor taken up in substantial amounts by the intestine (Table 3). Doing the same calculations as above, and neglecting the fact that some if not all of the glutamate released originated from glutamine (Table 3), it can be calculated that, if all the glutamine taken up were converted into glucose, this would have diluted the arterial glucose entering the intestinal circulation by 0.53% (0.085:2:8.00); taking into account that about half of this glutamine was converted into CO 2 , the corresponding value would have been only 0.27%. These values are different from that (7.9%) published by Mithieux and co-workers in 72h-fasted Sprague-Dawley rats [START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF]. Therefore our results clearly indicate that both in 72h-fasted Wistar rats and in 72hfasted Sprague-Dawley rats, glutamine gluconeogenesis cannot be detected in a reliable manner in the intestine in vivo. Since our data (Table 3) together with those of Windmueller and Spaeth [START_REF] Windmueller | Uptake and metabolism of plasma glutamine by the small intestine[END_REF] indicate that there were no other potential gluconeogenic precursors taken up by the intestine, this means that intestinal gluconeogenesis cannot be detected in vivo. In this context, the reasons why Windmueller and Spaeth [START_REF] Windmueller | Metabolism of absorbed aspartate, asparagine, and arginine by rat small intestine in vivo[END_REF][START_REF] Windmueller | Identification of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestine[END_REF] observed that small percentages of U-14 C-glutamine (4.7%), U-14 Clactate (0.1-0.2%) and U-14 C-aspartate (10%) were found in 14 C-glucose in the small intestine of their animals deserve careful examination. Since these authors had to chromatographically separate the various products resulting from the intestinal metabolism of their 14 C-labelled substrates, their 14 C-glucose fractions might have been contaminated by some other neutral 14 C-labelled compounds.

In this respect, we used 13 C NMR spectroscopy which avoids metabolite separations. In agreement Copyright 2006 Biochemical Society with this view, Windmueller and Spaeth [START_REF] Windmueller | Identification of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestine[END_REF] found that 6.2% of 14 C-D-β-hydroxybutyrate was also converted into 14 C-glucose; since ketone bodies cannot be gluconeogenic [START_REF] Krebs | The fate of isotopic carbon in kidney cortex synthesizing glucose from lactate[END_REF] their observation must have been due to some methodological artifact if one accepts that the small intestine does not synthesize glucose at all.

Reasons for the discrepancy between our in vivo results and those of Mithieux and co-workers

First, it should be pointed out that the arteriovenous difference approach has many limitations. Indeed, numerous experimental steps are necessary to get the final values [blood sampling, centrifugation to get the plasma, possible heterogeneity between the plasma and whole blood often neglected, deproteinization and neutralization of the sample, errors inherent to the enzymatic measurements of metabolites, imperfect chromatographic separation of radioactive metabolites, high blood concentration of glucose, radioactive measurements (radioactive disintegration is a random process), small arteriovenous differences multiplied by high blood flow sometimes measured in separate experiments]. Since each step has its own margin of error, the final values may suffer from substantial errors. Bearing this in mind, we believe that the gluconeogenic rates reported by Mithieux and co-workers in their 72h-fasted Sprague-Dawley rats [START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF] are not compatible with the possible uptake of gluconeogenic substrates. Indeed, they calculated an intestinal gluconeogenesis of 16.9 µmol.kg body wt -1 .min -1 . Taking a mean body weight of 270g and an intestinal blood flow of 5.2 ml.min -1 [START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF], this would mean that 0.885 (4.6: 5.2) µmole of glucose was added to each ml of blood passing through the intestine. Since two molecules of glutamine are needed to synthesize one molecule of glucose, their results would imply that 1.77 µmol of glutamine (or of other gluconeogenic precursor) was taken up from each ml of arterial blood only for the synthesis of glucose and without any complete oxidation of glutamine into CO 2 . Our data, presented in Table 3, indicate that in fact only 0.085 µmol of glutamine was taken up from each ml of arterial blood in 72h-fasted Sprague-Dawley rats (Table 3). Therefore, compelling evidence is experimentally provided that the gluconeogenic rates reported by Mithieux and Copyright 2006 Biochemical Society with the uptake and availability of glutamine nor with the availability and uptake of other circulating gluconeogenic precursors.

Moreover, taking into account that for each glucose synthesized two glutamine carbon skeletons should pass through phosphoenolpyruvate carboxykinase, a mandatory enzyme in glutamine gluconeogenesis, it is important to note that the Vmax of phosphoenolpyruvate carboxykinase measured by Mithieux et al. was severalfold lower than the rates of gluconeogenesis they calculated [START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF]. This represents another compelling piece of evidence that the high gluconeogenic rates reported by the latter authors are not compatible with the biochemical characteristics of the intestine of their 72h-fasted Sprague-Dawley rats.

Glycogen stores and glucose release in vivo

Our measurements indicate that the glycogen stores in the entire small intestine of 72h-fasted Wistar rats were 13 µmoles (in glucosyl equivalents). The complete release of this glycogen as glucose via glycogenolysis would have to occur within 3 minutes to account for the rate of release of newly synthesized glucose reported by Mithieux and co-workers [START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF]. Thus, glycogen cannot be the source of the glucose calculated to be released in vivo by these authors.

Ketone body metabolism

Another interesting observation made in the present study is that, in the 72h-fasted rat, there were major strain differences that, to our knowledge, have never been reported so far. Indeed, the intestine of 72h-fasted Sprague-Dawley rats did not remove circulating ketone bodies at all. By contrast, the intestine of 72h-fasted Wistar rats removed circulating D-β-hydroxybutyrate but released about 60% of it as acetoacetate. Thus, these two strains of rats differ from the Mendel-Osborne rats used by Windmueller and Spaeth who found that, after an overnight fast, the intestine took up both D-βhydroxybutyrate and acetoacetate [START_REF] Windmueller | Identification of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestine[END_REF]. Therefore, the traditional view that ketone bodies are important energy substrates of the intestine in the fasting state does not necessarily apply to all strains of rats. In summary, our in vitro data clearly demonstrate that metabolically viable segments from the small intestine of 72h-fasted rats did not synthesize glucose at all despite a high rate of glutamine utilization and metabolism. Our in vivo data not only in Wistar but also in Sprague-Dawley rats after 72h of fasting indicate that the rate of intestinal gluconeogenesis reported by Mithieux et al. [START_REF] Mithieux | Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes[END_REF] in their 72h-fasted Sprague-Dawley rats can be accounted for neither by the uptake of the gluconeogenic precursors available in the arterial blood, nor by the Vmax of phosphoenolpyruvate carboxykinase they measured. Moreover, our results demonstrate that glutamine gluconeogenesis would not be detectable by the currently available methods because the dilution of circulating glucose by newly synthesized glucose would be much smaller than the margin of errors of the methods employed. Therefore, the view that the intestine of the rat is an important site of gluconeogenesis, at least in 72h-fasted rats, is not tenable anymore. Intestinal segments (267 ± 14 mg protein per flask) were incubated for 20 and 30 min as described in the Methods section. Results for metabolite removal (-)

or production (measured enzymatically and given in µmol.g protein -1 .incubation time -1 ) are reported as means ± S.E.M. for four experiments. The 13 C NMR data corresponding to these experiments are reported in Table 2. 13 C]glutamine in the absence (Fig. 1) and the presence (Fig. 2) of glucosamine.

  20 and 30 min) in 250 ml Erlenmeyer flasks containing an atmosphere of O 2 /CO 2 (19:1) and 20 ml of Krebs-Henseleit buffer without or with 5mM L-[3-13 C]glutamine, 1 or 5 mM unlabelled or D-[2-Biochemical Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148 Copyright 2006 Biochemical Society

Figure 1

 1 Figure1shows a representative13 C NMR spectrum of perchloric extracts obtained after 30 min of incubation of intestinal segments from a 72h-fasted rat with 5 mM [3-13 C]glutamine. This spectrum,

  and 2), clearly demonstrates the presence of glucose carbon resonances. However, as suggested by the fact that all the carbon resonances (the sums of the anomers α and β) had virtually the same intensities (peak heights) and peak areas, these resonances corresponded exclusively to the natural abundance of the glucose carbons added at zero-time and not metabolized by the intestinal segments after 30 min of Biochemical Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148 Copyright 2006 Biochemical Society incubation. Therefore, no synthesis of 13 C-labelled glucose carbons occurred from [3-13 C]glutamine in these experiments.

Biochemical

  Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148 Copyright 2006 Biochemical Society metabolized glutamine avidly and in a virtually linear manner with time (Tables

Biochemical

  Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148

Fig. 3 .

 3 Fig. 3. 13 C NMR spectrum (125.17 MHz) of neutralized perchloric acid extracts obtained from

33

 33

  Figure 2

  34

Figure 3

 3 Figure 3

Biochemical Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148 Copyright 2006 Biochemical Society

Biochemical Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148

Biochemical Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148

  C]glucose measured by NMR spectroscopy; the corresponding values were 55.7 ± 1.5 and 36.6 ± 1.8

Biochemical Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148

Biochemical Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148

Biochemical Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148

  co-workers are compatible neither

Table 1 . Metabolism of 5 mM L-[3-13 C]glutamine in small intestinal segments from 72h-fasted rats.

 1

	Metabolite removal (-) or production

Table 3 . Arterial and portal vein blood concentrations of metabolites in 72h-fasted Wistar and Sprague-Dawley rats.

 3 of rats. The metabolites were measured as described in the Methods section. Values are means ± S.E.M. Statistical difference was measured by the paired Student's t test for paired data between the arterial and portal vein blood: * P<0.05.

	Blood metabolite concentration

Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148 Copyright 2006 Biochemical Society Legends to figures Figs. 1 and 2. 13 C NMR spectra (125.17 MHz) of neutralized perchloric acid extracts obtained from small intestinal segments (from 72h-fasted Wistar rats) incubated during 30 min with [3-

  1, alanine C-3 (17.42); 2, lactate C-3 (21.45); 3, unidentified and unmetabolized glutamine C-3 contaminant (26.41); 4, glutamine C-3 (27.06 & 27.20 & 27.33); 5 glutamate C-3 (28.01 & 27.88 & 27.74); 6, ornithine C-3 (28.41); 7, citrulline C-3 (28.77); 8, proline C-3 (30.07); 9, glutamine C-4 (31.88 & 32.16); 10, glutamate C-4 (34.88); 11, aspartate C-3 (37.57); 12, unidentified (38.57); 13, glycine C-2 (42.48 & 42.70 & 42.91) added as internal standard; 14, unidentified (48.52); 15, artifact generated by the spectrometer in the presence of Krebs-Henseleit buffer and/or biological samples (50.18); 16, alanine C-2 (51.64); 17, aspartate C-2 (53.09); 18, glutamine C-2 (55.10 & 55.38); 19, glutamate C-2 (55.62); 20, lactate C-2 (69.51); 21, glycine C-1 (173.49 & 173.70 & 173.91); 22, glutamine C-1 (175.23); 23, glutamate C-1 (175.71); 24, alanine C-1 (177.03); 25, glutamine C-5 (178.62); 26, glutamate C-5 (182.48); 27, lactate C-1 (183.70). Glucosamine resonances are indicated by the uppercase letter "G". The chemical shifts are: 55.28 ; 57.80 ; 61.43; 61.56; 63.45; 63.96; 70.54; 70.67; 72.03; 72.06; 72.61; 72.88; 74.39; 75.31; 77.05; 90.11; 93.63; 143.07; 143.20; 145.36; 154.22; 154.75; 155.88.

C]glucose. Glucosamine (40 mM), an inhibitor of hexokinase[START_REF] Guglielminetti | Characterization of isoforms of hexose kinases in rice embryo[END_REF], was also used in certain

Acknowledgements

We thank Claire Morel for secretarial assistance. Biochemical Journal Immediate Publication. Published on 27 Sep 2006 as manuscript BJ20061148 Copyright 2006 Biochemical Society