Javier Pizarro-Delgado

Inés Hernández-Fisac

Rafael Martín-Del-Río

Jorge Tamarit-Rodriguez

Jorge Tamarit-Rodríguez

BRANCHED-CHAIN 2-OXOACIDS TRANSAMINATION INCREASES "GABA-SHUNT" METABOLISM AND INSULIN SECRETION IN ISOLATED ISLETS Short title: Branched-chain 2-oxoacids' transamination increase islet GABA metabolism and insulin secretion

Keywords: Islets, insulin, GABA, branched-chain 2-oxoacids' transaminase, GABA transaminase, GABA metabolism

BCAT c and BCAT m (branched-chain amino acid transferase, cytosolic and mitochondrial isozymes, respectively); 2OGdh (2-oxoglutarate dehydrogenase); GDH (glutamic acid dehydrogenase); GAD (glutamic acid decarboxylase); GABA-T (GABA transaminase); SSA-R (semialdehyde succinic acid reductase); SSA-dh (semialdehyde succinic acid dehydrogenase); MDH (malic enzyme); BCKA (branchedchain 2-oxoacids): KIC (oxo-4-methylpentanoate or α-ketoisocaproate), KMV (oxo-3methylpentanoate or α-keto-β-methylpentanoate), KC (oxohexanoate or α

INTRODUCTION

Branched-chain 2-oxoacids (also known as branched-chain α-keto acids, BCKA) trigger a biphasic secretion of insulin in perifused rat islets which is similar in form to that stimulated by high glucose [START_REF] Panten | Effects of L-leucine and α-ketoisocaproic acid upon insulin secretion and metabolism of isolated pancreatic islets[END_REF][START_REF] Panten | Effects of alpha-ketomonocarboxylic acids upon insulin secretion and metabolism of isolated pancreatic islets[END_REF][START_REF] Lenzen | Effects of α-ketocarboxylic acids and 4-pentenoic acid on insulin secretion from the perfused rat pancreas[END_REF][START_REF] Lenzen | 2-oxocarboxylic acids and function of pancreatic islets in obese-hyperglycaemic mice[END_REF]. Early studies showed that BCKA were transaminated in a reaction probably catalyzed by a specific branched-chain 2-oxoacid aminotransferase (BCAT) activity [START_REF] Hutton | The metabolism of 4-methyl-2oxopentanoate in rat pancreatic islets[END_REF][START_REF] Malaisse | The stimulus-secretion coupling of amino acid-induced insulin release. Metabolic interaction of L-glutamine and 2-ketoisocaproate in pancreatic islets[END_REF][START_REF] Lenzen | Transamination of neutral amino acids in pancreatic B-cell mitochondria[END_REF][START_REF] Lenzen | 2-Ketoglutarate generation in pancreatic B-cell mitochondria regulates insulin secretory action of amino acids and 2-keto acids[END_REF]. Moreover, they can be oxidised to CO 2 an acetoacetate [START_REF] Hutton | The metabolism of 4-methyl-2oxopentanoate in rat pancreatic islets[END_REF][START_REF] Panten | Recognition of insulin-releasing fuels by pancreatic B-cells. α-Ketoisocaproic acid is an appropriate model compound to study the role of β-cell metabolism[END_REF][START_REF] Lenzen | Signal function of metabolism of neutral amino acids and 2-keto acids for initiation of insulin secretion[END_REF] and it has been shown that oxo-4-methylpentanoate (also known as α-ketoisocaproate, KIC) is oxidized at a similar rate to high glucose and its stimulation of insulin secretion might share a common mechanism with the sugar, consisting of an increased catabolic rate [START_REF] Hutton | Similarities in the stimulussecretion coupling mechanisms of stimulation of glucose-and 2-keto acidinduced insulin release[END_REF]. As BCKA may generate 2-oxoglutarate (2OG) in their transamination reaction with glutamate [START_REF] Lenzen | 2-Ketoglutarate generation in pancreatic B-cell mitochondria regulates insulin secretory action of amino acids and 2-keto acids[END_REF] and hence feed the tricarboxylic acid cycle, their different secretory capacities might also depend on their different rates of transamination. However, this turned out not to be the case and oxo-3-methylbutyrate (also known as α-ketoisovalerate, KIV), which does not stimulate insulin secretion, showed the maximum rate of transamination in islet homogenates [START_REF] Malaisse | The stimulus-secretion coupling of amino acid-induced insulin release. Metabolic interaction of L-glutamine and 2-ketoisocaproate in pancreatic islets[END_REF]. Since it was found that L-leucine activates glutamate dehydrogenase (GDH) [START_REF] Sener | L-leucine and a nonmetabolized analogue activate pancreatic islet glutamate dehydrogenase[END_REF], KIC transamination gained importance as a putative mechanism for increasing energy production and hence insulin release [START_REF] Lenzen | Transamination of neutral amino acids in pancreatic B-cell mitochondria[END_REF]. However, most of the branched chain α-amino acids (Val, Ileu, norLeu), except L-leucine, are not GDH-activators even though some of their corresponding 2-oxocarboxylic acids may trigger a biphasic insulin secretion (oxo-3-methylpentanoate and oxohexanoate, also known as α-keto-β-methyl valeric acid or KMV and α-ketocaproic acid or KC, respectively) [START_REF] Lenzen | 2-Ketoglutarate generation in pancreatic B-cell mitochondria regulates insulin secretory action of amino acids and 2-keto acids[END_REF]. It has therefore not yet been settled which is the main metabolic process contributing to the stimulation of insulin secretion by BCKA in β-cells: their increased catabolism (oxidation), their transamination rate and the generation of 2OG, and/or the stimulation of GDH.

BCAT activity is present as two isoenzymes in the brain: a cytosolic one (BCATc) predominates in neurones whereas a mitochondrial form (BCATm) is more generally expressed in astrocytes and peripheral tissues [START_REF] Goto | Structural determinants for branched-chain aminotransferase isozyme-specific inhibition by the anticonvulsant drug gabapentin[END_REF]. Both enzyme activities have been demonstrated in islets of ob/ob mice and no preference for any of the two was found for the BCKA secretagogues KIC or KC [START_REF] Lenzen | Transamination of neutral amino acids in pancreatic B-cell mitochondria[END_REF]. The neuroactive drug gabapentin (1-(aminomethyl)cyclohexaneacetic acid) is a rather specific competitive inhibitor of BCAT-c with a Ki (0.8-1.4 mmol/l) value similar to the Km for L-leucine whereas much higher concentrations (65,4 mmol/l ) are required to inhibit L-leucine binding to BCATm [START_REF] Goldlust | Effects of anticonvulsant drug gabapentin on the enzymes in metabolic pathways of glutamate and GABA[END_REF]. This drug, in combination with others, will be used to characterize the isozyme pattern of BCKA transamination in an attempt to explore its relationship with the capacity to stimulate insulin secretion.

KIC has also been found to be capable of significantly reducing intracellular GABA levels in rat islets [START_REF] Hernández-Fisac | Oxo-4-methylpentanoic acid directs the metabolism of GABA into the Krebs Cycle in rat pancreatic islets[END_REF]. As the rate of GABA release was not modified by KIC and no intracellular accumulation of L-leucine could be demonstrated (it accumulated extracellularly), it was proposed that the 2OG generated in the transamination reaction might feed GABA-transaminase activity and promote metabolism of the amine in the GABA-shunt [START_REF] Hernández-Fisac | Oxo-4-methylpentanoic acid directs the metabolism of GABA into the Krebs Cycle in rat pancreatic islets[END_REF]. BCKA are known to inhibit the mitochondrial metabolism of 2OG [START_REF] Shestopalov | Branched-chain keto-acids exert biphasic effects on α-ketoglutarate-stimulated respiration in intact rat liver mitochondria[END_REF] as well as 2-oxoglutarate dehydrogenase (2OGdh) activity [START_REF] Patel | Inhibition by the branched-chain 2-oxo acids of the 2oxoglutarate dehydrogenase complex in developing rat and human brain[END_REF]. If this could be demonstrated in islet cells, the idea that BCKA stimulate insulin secretion by increasing "GABA-shunt" metabolism would be reinforced. Therefore, the aim of this study was to investigate the dependence of BCKA capacity to stimulate insulin secretion and to decrease islet GABA on their rate of transamination using BCAT inhibitors.

Methods

Islets were isolated from the pancreas of male Wistar-albino rats (250-275 g BW) by collagenase digestion. Insulin secretion was studied in perifused or incubated islets. Two groups, each of 40 rat islets, were perifused in parallel and at a flow rate of 0.5 ml/min with Krebs-Ringer, buffered with 0.5 mM NaHCO 3 and 20 mM HEPES, supplemented with 0.5 % FFA-free BSA (KRBH), and heated at 37 0 C. The perifusion pattern was similar in all the experiments. After a pre-perifusion period of 45 minutes under basal conditions (in the absence of nutrients or at non-stimulatory concentrations of substrates), the perifusion medium was switched to one containing the test substances and maintained for the next 30 minutes. Finally, the medium was changed back to preperifusion conditions where it was maintained for the last 25 minutes. The perifusate was collected at 1 minute intervals during the last 60-70 minutes of perifusion and its insulin concentration radioimmunologically measured. Alternatively, three batches each of ten islets were incubated in 1 ml of KRBH at 37 0 C for 60 minutes and the concentration of insulin released in the incubation medium was radioimmunologically measured. Pig insulin was radio-iodinated with Na 125 I [START_REF] Hunter | Preparation of iodine-131 labeled human growth hormone of high specific activity[END_REF] and rat insulin was used as a standard in the radioimmunoassay of insulin. Insulin antiserum was kindly provided by Dr. Janove Sehlin from the Department of Medical Cell Biology at the University of Umeå (Sweden).

Islet α-amino acids were separated by reverse-phase HPLC after precolumn derivatization with o-phthalaldialdehyde [START_REF] Jones | Amino acid analysis and enzymatic sequence determination of peptides by an improved o-phtalaldialdehyde precolumn labeling procedure[END_REF] and quantified by fluorescence detection. Islets (groups of 20) were incubated for 60 minutes at 37 0 C in 70 µl of the same buffer as that used in perifusions. Incubations were stopped by placing the tubes containing the islets on ice. The medium was aspirated and the islets were washed twice with 100 µl of cold PBS. Finally, 30 µl of 10 % (w/v) 5-sulfosalicylic acid were added in order to extract islet amino acids. Final extracts were kept at -40 0 C until their amino acid content was determined. Twelve amino acids were regularly identified and separated in the extracts and they were measured with a sensitivity close to 1 pmol (Asp, Glu, Ser, Gln, His, Gly, Thr, Arg, Tau, Ala, Tyr, and GABA). Occasionally, the gradient elution profile of the chromatographic separation was changed in order to separate and quantify also branched-chain α-amino acids (Leu, Ileu, Val). Islet DNA was measured with a fluorometric method [START_REF] Vytasek | A sensitive assay for the determination of DNA[END_REF] in an extract of islets that had been previously sonicated (four times, ten strokes at 10 % of the cycle and the minimum potency of a Branson sonifier 450) in 100 µl 5 mmol/l NaOH. In experiments aimed at investigating the release of amino acids, islets (groups of 30) were incubated for 60 minutes at 37 0 C in 70 µl of the same buffer as that used in perifusions. After stopping the incubation on ice, 50 µl of the supernatant was withdrawn and stored frozen until amino acid measurement was A c c e p t e d M a n u s c r i p t performed. The remaining medium was discarded and the islets were processed as described before for the measurement of their amino acid content. 2OGdh activity in islets' homogenates was studied as the amount of NADH produced at saturating concentrations of 2OG (5 mM) and NAD + (2.5 mM) and measured fluorometrically in a multiwell plate (Costar assay plate no. 3915, Corning Incorporated, NY, U.S.A.) recorder (Varioskan model, Bioanalysis Labsystems S.A., Barcelona, Spain). Isolated islets were homogenized by sonication (four times, ten strokes at 10-20 % of the cycle and the minimum potency of a Branson sonifier 450) in a buffer (10 mM KH 2 PO 4 and 0.5 mM EDTA, pH= 7.2) containing: leupeptin (60 µM), aprotinin (0.0188 TIU/ml), PMSF (2 mM in DMSO), β-mercaptoethanol (10 mM), and Triton-X100 (0.5 %, vol/vol). The islet homogenate was then centrifuged at 4 0 C and 141,500 g for 30 minutes in a 100.Ti rotor of the ultracentrifuge model Optima TM -L-100 XP (Beckman Coulter España S.A.). The enzyme reaction was performed in the same buffer supplemented with 2 mM oxamate, 4 µM rotenone, 0.5 mM coenzyme A, 0.3 mM thiamine pyrophosphate, 2.5 mM dithiotreitol, 0.8 mM CaCl 2 , 1 mM MgCl, 5 mg/ml BSA and the substrates. The enzyme activity was calculated as the slope of the linear increase of NADH fluorescence expressed as the amount of formed product / min x mg protein by reference to a nucleotide standard curve. The amount of protein was measured in the islet extracts with the method of Lowry [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF] and BSA was used as the standard.

Branched-chain 2-oxoacid aminotransferase activity (BCAT) was measured in islets' homogenates as the amount of the branched-chain α-amino acid (Leu, Ileu, nor-Leu or Val) produced at saturating concentrations (1 mM) of the branched-chain α-keto acid (KIC, KMV, KC or KIV, respectively) and L-glutamate (2 mM). Each branched-chain α-amino acid was isocratically separated by reverse-phase HPLC and fluorometrically detected after its derivatization with o-phtaladialdehyde, as described above for the measurements of islet α-amino acids. The reaction was linear for at least 20 minutes and the amount of α-amino acid accumulated during this time period was used to calculate enzyme activity. Isolated islets were homogenized by sonication (four times, ten strokes at 10 % of the cycle and the minimum potency of a Branson sonifier 450) in a buffer (50 mM KH 2 PO 4 and 1.0 mM EDTA, pH= 7.8) containing: leupeptin (60 µM), aprotinin (0.0188 TIU/ml), PMSF (2 mM in DMSO), β-mercaptoethanol (1.4 mM), and Triton-X100 (0.5 %, vol/vol). The islet homogenate was then centrifuged at 4 0 C and 141,500 g for 30 minutes in a 100.Ti rotor of an ultracentrifuge (model Optima TM -L-100 XP, Beckman Coulter España S.A.). The enzyme reaction was performed in the same buffer supplemented with 50 µM pyridoxal phosphate and the two substrates.

All the experimental data are here presented as the mean values ± SE, and the numbers of separate experiments are given in parentheses. Statistical comparisons were performed with non-paired, two-tailed Student's t tests.

RESULTS

Effect of BCAT inhibitors on insulin secretion.

BCKA showed different potencies of stimulation of insulin secretion when tested on perifused islets at 10 mM (Fig. 1). KIC and KC stimulated a similar, biphasic, secretory response that was slightly higher than that triggered by KMV. KIV only induced a small first phase release and an even lower second phase. In a preliminary screening, at a fixed concentration, of the capacity of different BCAT inhibitors to modify BCKA- stimulation of insulin secretion, a greater sensitivity of KMV towards 10 mM DL-αmethylleucine (-35.3 ± 6.8 %,n=7; p<0.01) and 5 mM gabapentin (-60.4 ± 8.8 %, n=8; p< 0.0004) was observed, as compared with KIC, which was not affected at all. In fact, 5 mM gabapentin suppressed within 52% the second phase of KMV-induced insulin release without modifying the first phase (Fig 2). However, 10 mM 4-methylvaleric acid (MVA), a L-leucine structural analogue [START_REF] Goto | Structural determinants for branched-chain aminotransferase isozyme-specific inhibition by the anticonvulsant drug gabapentin[END_REF], was more effective than the other two inhibitors in the suppression of both KMV (-92.4 ± 1.6 %, n=8; p< 0.001) and KIC (-94.7 ± 0.5 %, n=8; p< 0.001) stimulation of insulin secretion.

As shown in Table 1, KMV and KIC exhibited a different sensitivity towards gabapentin-induced inhibition of their secretory response: whereas the response triggered by 10 mM KMV was significantly decreased by 10 (-37 %) and 50 mM (-59 %) gabapentin, 10 mM KIC-stimulation was resistant to 10 mM (-14 %) and it was suppressed, but to a lower degree, by 50 mM (-38 %) gabapentin. In batch-type incubations, KIV did not increase insulin secretion above the basal level, but its response was strongly potentiated by 10 mM L-glutamine (Table 1) and this potentiation was dose-dependently decreased by 10 (-57 %) and 50 mM (-77 %) gabapentin. MVA suppressed very efficiently, and dose-dependently, 10 mM KICinduced secretion, up to -90% at 20 mM (Table 1).

Islet viability after 30 minutes' (as in the perifusions) exposure to BCAT inhibitors was checked after washing the islets and changing the medium: Pre-treatment of islets with either 10 mM KIC alone or together with 50 mM gabapentin or 20 mM VMA did not modify their subsequent insulin secretory response to 20 mM glucose, nor their insulin content (results not shown).

BCKA transamination

BCKA transamination with L-glutamate was measured in islet homogenates measuring the linear accumulation of the resulting α-amino acid and the effect of a concentration range of different inhibitors investigated. A hierarchy of transamination activities was observed among the different BCKA assayed: the highest was obtained with KIV, followed in a decreasing order by KIC, KMV and KC (Table 2). KIV and KMV transaminations were more sensitive to the inhibitory effect of gabapentin than those of KIC and KC: whereas transamination of the two former were significantly decreased by 2 and 10 mM gabapentin, the inhibitor did not modify the transamination of the latter two. Moreover, 50 mM gabapentin caused a greater suppression of transamination with KIV (-85.5 %) and KMV (-78.3 %) than with either KIC (-61.0 %) and KC (-55.6 %). MVA behaved as a potent inhibitor of BCKA transamination (over 87% at 20 mM) and did not show any preference for a particular branched-chain α-keto acid (Table 2). DLα-methylleucine suppressed 10 mM KIC-transamination at 10 and 20 mM within 33.0 and 43.5 %, respectively (Table 2).

BCKA transamination activity was also measured in intact islets preloaded for 1 hour with various L-glutamine concentrations (0, 0.5 and 10.0 mM): the amount of α-amino acids, either released or remaining in the islets, was measured after washing the islets (to rid them of Gln) and incubating them for a second hour in the presence of a 10 mM concentration of the different BCKAs. No significant accumulation of any of the branched chain α-amino acids (Leu, Ileu or Val) could be detected in the islets at any Lglutamine concentration (results not shown). As shown in Table 3, the amount of the branched-chain α-amino acid released (in vivo transamination) increased with the concentration of L-glutamine to which islets were pre-exposed during the first hour. In the absence of L-glutamine, the production of Leu was significantly greater than that of Ileu but this difference disappeared at either 0.5 or 10.0 mM L-glutamine. By contrast, the amount of Val released was consistently lower than that of Leu at all the Lglutamine concentrations tested (Table 3). Both, KMV and KIV, significantly increased the islet content of Asp and Glu with respect to the control and also in comparison to KIC (Table 3). KIC was the only BCKA that significantly decreased islet GABA content in the absence of L-glutamine (Table 3). However, at a L-glutamine concentration close to the physiological plasma concentration (0.5 mM) or higher (10 mM), all three BCKA significantly diminished the islet GABA content.

In order to check the hypothesis that BCKA transamination is responsible for increased GABA metabolism and therefore for the stimulation of insulin secretion, we investigated whether the inhibition of transamination would also block the reduction of islet GABA. For that purpose, MVA was used as the BCAT inhibitor because it suppressed with similar efficiency KIC-induced insulin secretion and the rate of α-keto acid transamination to Leu in islet homogenates. The effect of MVA, measured with the same experimental design used to evaluate in vivo transamination (see preceding paragraph), was tested on islets that had been pre-loaded for 1 hour with 10 mM Lglutamine. 10 mM MVA suppressed within 67% the release of Leu (4.0 ± 0.4, n=8 vs. 1.3 ± 0.2 pmoles/ng DNA, n=8; p<0.0001) evidencing its efficient inhibition of KIC transamination in intact islets. In contrast, it did not modify the release of any other αamino acid (results not shown). Simultaneously, MVA increased by 57 % the islet GABA content in the presence of 10 mM KIC ( 3.7 ± 0.5, n= 8 vs. 2.4 ± 0.2 pmoles/ng DNA, n=8; p< 0.028) and induced a parallel decrease of Asp (1.3 ± 0.2, n=8 vs. 2.3 ± 0.2 pmoles/ng DNA, n=8; p<0.001) and Glu (0.7 ± 0.06, n=8 vs. 1.3 ± 0.2 pmoles/ng DNA; p<0.008). The islet content of Leu was not modified at all (0.19 ± 0.02, n=8 vs. 0.18 ± 0.04 pmoles/ng DNA, n=8; N.S.). The membrane permeable GABA analogue ethyl-GABA (eGABA) increased the islet content of GABA in the presence of 10 mM KIC by 66 % at 1.5 mM (3.9 ± 0.4, n=8 vs. 2.4 ± 0.2 pmoles/ng DNA, n=8; p<0.002) and elevated it three-fold at 5 mM (7.0 ± 0.7, n=8 vs. 2.4 ± 0.2 pmoles/ng; p< 0.002). Neither 1.5 nor 5.0 mM eGABA modified the rate of KIC transamination measured as Leu release (4.2 ± 0.4, n=8 and 4.0 ± 0.3, n=8 vs. 4.0 ± 0.6 pmoles/ng DNA, n=8).

Effect of the intracellular GABA concentration on insulin secretion.

The islet GABA content is dose-dependently increased by the extracellular L-glutamine concentration and reaches close to the maximum level at the physiological plasma concentration of the α-amino acid (0.5 to 1.0 mM) [START_REF] Fernández-Pascual | Conversión into GABA (γ-aminobutyric acid) may reduce the capacity of L-glutamine as an insulin secretagogue[END_REF]. We have shown before [START_REF] Hernández-Fisac | Oxo-4-methylpentanoic acid directs the metabolism of GABA into the Krebs Cycle in rat pancreatic islets[END_REF], and corroborated in this work (Table 3), that KIC efficiently reduces the islet content of GABA at any L-glutamine concentration. This effect was ascribed to 2OG generation in the transamination of KIC to Leu and the subsequent GABA transamination with the generated 2OG.

We showed above that increasing the intracellular GABA concentration at two different levels with eGABA (1.5 and 5.0 mM) did not modify the rate of KIC transamination measured as the release of Leu. This apparently suggests that the observed suppression of intracellular GABA does not limit the rate of KIC transamination but perhaps restricts GABA transamination. We therefore explored whether the secretory response to KIC might be improved by increasing intracellular GABA levels with the membrane permeable analogue. As shown in Fig 3A, the simultaneous perifusion of rat islets with 10 mM KIC together with 1 mM eGABA did not modify the first phase of insulin secretion but strongly decreased the sustained, second phase of insulin release by more than 50 % (16.2 ± 3.0, n= 4, vs. 33.5 ± 3.0 ng insulin/ 20min x 40 islets, n= 6, p<0.005). A further increase of intracellular GABA with 5 mM eGABA decreased slightly more (-77%) the second phase of KIC-induced insulin secretion (5.4 ± 1.4, n= 5, vs. 23.3 ± 4.4 ng insulin/ 20min x 40 islets, n= 4; p< 0.002) but did not modify first phase release (results not shown). The second phase of the secretory response to 10 mM KMV was similarly decreased by 5 mM eGABA within 86 % (4.5 ± 1.7, n=6, vs. 32.0 ± 5.3 ng insulin/ 20min x 40 islets, n= 4; p<0.0004) (Fig. 3B). These results suggest that islet GABA, besides being a potential metabolic substrate, might have other effects on the mechanism of insulin secretion above certain concentrations.

Preliminary results have shown that 5 mM eGABA significantly decreases glucoseinduced insulin secretion and this effect correlated with a hyperpolarization of the β-cell plasma membrane [START_REF] Tamarit-Rodriguez | Intracellular GABA accumulation suppresses glucose-induced insulin secretion in rat pancreatic islets as well as membrane depolarization in β cells[END_REF]. Therefore, we tried to counteract eGABA-induced decrease of the secretory response to KIC by depolarizing the islets with 70 mM KCl: surprisingly, depolarization itself suppressed the second phase of release within 61% (8.8 ± 0.8, n= 27 vs. 22.6 ± 1.7 ng insulin/ 20min x 40 islets, n= 27; p<0.0001) (Fig. 4) whereas it had no effect on glucose (20 mM) stimulation (results not shown). The addition of 1, 5 or 10 mM eGABA did not modify depolarization induced suppression of the second phase of release triggered by 10 mM KIC (results not shown). A smaller reduction of the K +electrochemical potential with 30 mM KCl did not modify the secretory response (results not shown). Whereas 70 mM KCl depolarization did not significantly affect KMV-induced release (12.3 ± 1.9, n=4, vs. 20.8 ± 3.8 ng insulin/ 20min x 40 islets, n=4; N.S.) (Fig. 5A), it suppressed by 63 % the second phase of secretion triggered by 10 mM KC (9.9 ± 1.3, n=6, vs. 27.2 ± 0.8 ng insulin/ 20 min x 40 islets, n=5; p<0.0001) (Fig 5B).

As we have hypothesized [START_REF] Hernández-Fisac | Oxo-4-methylpentanoic acid directs the metabolism of GABA into the Krebs Cycle in rat pancreatic islets[END_REF] that KIC stimulation of insulin secretion is at least partially dependent on the promotion of GABA metabolism, we checked whether the decrease of the K + -electrochemical potential by 70 mM KCl might interfere with the availability of GABA in the islets. For that purpose, islets were pre-loaded with 10 mM L-glutamine for 1 hour and, after washing the remaining extracellular Gln, both islet content and the release of α-amino acids were measured after a second hour of incubation in the presence of 70 mM KCl. Table 4 shows that under this depolarizing condition, the release of GABA and taurine were increased by more than 100 % and this increment was not affected by the simultaneous presence of 10 mM KIC. There were corresponding decreases of both GABA and Tau contents (-32 % and -40 %, respectively). It was shown again that 10 mM KIC alone decreased within 42 % islet GABA content without modifying Tau content. KIC was even capable of suppressing further GABA (p<0.008), but not taurine, content in the presence of 70 mM KCl.

Modification of 2-oxoglutarate dehydrogenase (2OGdh) activity by BCKA in islet homogenates.

This enzymatic activity competes for its substrate with GABA transaminase, the enzyme that initiates metabolism in the "GABA-shunt". The confirmation in islet homogenates that 2OGdh activity is inhibited by BCKAs, as has been demonstrated in other tissues [START_REF] Shestopalov | Branched-chain keto-acids exert biphasic effects on α-ketoglutarate-stimulated respiration in intact rat liver mitochondria[END_REF][START_REF] Patel | Inhibition by the branched-chain 2-oxo acids of the 2oxoglutarate dehydrogenase complex in developing rat and human brain[END_REF], would strengthen the importance of an increased "GABA-shunt" metabolism in the stimulation of insulin secretion by KIC. As shown in Table 5, KIC decreased 2OGdh activity in islet homogenates in a dose-responsive manner between 1 and 10 mmol/l. At 10 mM, KIC decreased the enzyme activity to around 6 % of the control. Similar effects were found in homogenates of rat cerebellum (not shown). 5 mM KMV induced a stronger reduction of 2OGdh activity than 5 mM KIC in both islets

DISCUSSION

It has been shown recently that the secretory response to KIC in incubated mouse islets was inhibited by DL-α-methylleucine in a concentration dependent manner although the drug effect on islet transamination was not checked [START_REF] Rabaglia | α-Ketoisocaproate-induced hypersecretion of insulin by islets from diabetes-susceptible mice[END_REF]. We have demonstrated in this paper that DL-α-methylleucine inhibited KIC transamination in rat islet homogenates in a concentration sensitive manner but that it did not suppress KIC-induced stimulation of insulin secretion. This different sensitivity to DL-methylleucine between mouse [START_REF] Rabaglia | α-Ketoisocaproate-induced hypersecretion of insulin by islets from diabetes-susceptible mice[END_REF] and rat islets might be attributed to the species differences. Gabapentin is a well characterized and specific inhibitor of the cytosolic form of BCAT which is known to predominate in neurones [START_REF] Goto | Structural determinants for branched-chain aminotransferase isozyme-specific inhibition by the anticonvulsant drug gabapentin[END_REF]. Therefore, it was used in this study as a tool to uncover specific differences in the affinity among the different BCKA for the transaminase isoforms. Our results confirm previous evidence indicating that both cytosolic and mitochondrial BCAT activities are present in rat islets [START_REF] Lenzen | Transamination of neutral amino acids in pancreatic B-cell mitochondria[END_REF]. The study of the effect of a range of gabapentin concentrations on BCKA transamination in islet homogenates allows us to conclude that both KIC and KC, at variance with KMV and KIV, have a higher affinity for the mitochondrial than the cytosolic isoform. The existence of a mitochondrial activity might facilitate the concatenation of 2-oxoacid transaminase and GABA transaminase activities when islets are exposed to KIC and KC (see diagram in Figure 6). This would result in a greater efficiency of KIC and KC to reduce intracellular GABA compared to KMV and KIV which are preferentially transaminated by the cytosolic isoenzyme. This was confirmed for KIC in the present study. In previous experiments in our laboratory, it was found that KC induced a greater decrease of islet GABA (-53%) than KMV (-32 %) in the absence of Gln (results not shown).

The weak capacity of KIV to decrease intracellular GABA correlates with its low secretory competence and is in apparent contradiction with its high rate of transamination in islets' homogenates. The rank of transamination activities found in this work (KIV>KIC>KMV>KC) confirms previously published results [START_REF] Malaisse | The stimulus-secretion coupling of amino acid-induced insulin release. Metabolic interaction of L-glutamine and 2-ketoisocaproate in pancreatic islets[END_REF] in rat islets and emphasizes the lack of correlation between transamination rate and secretory strength. However, when tested in vivo, KIV conversion to Val was much lower than synthesis and release of Leu from KIC under all Gln concentrations studied; KMV transamination to Ileu was also lower than Leu formation from KIC. These differences in transamination rates may be, at least partially, determined by differences in transport rates through the plasma membrane of β-cells. They correlate better than in vitro transamination rates with the secretory potency of the different BCKA and their capacity to decrease islet GABA. In conclusion, the importance of BCKA transamination in their mechanism of stimulation of insulin secretion now seems to be settled. As a corollary, BCKA oxidation does not seem to contribute much to the stimulation of secretion, as pointed out by Rabaglia et al [START_REF] Rabaglia | α-Ketoisocaproate-induced hypersecretion of insulin by islets from diabetes-susceptible mice[END_REF]. Moreover, it has been shown that KIC by itself is not a good substrate in isolated mitochondria but it increases ATP production in combination with L-glutamate (25). This supports our finding that KIC is mainly transaminated by the mitochondrial form of BCAT in islet cells.

If BCKA oxidation is not important, what would then be the source of metabolic energy used to initiate and maintain insulin secretion? A "non metabolic" decrease of islet GABA induced by its increased release after high K + (70 mM KCl) depolarization (Table 4) significantly suppressed the second phase of insulin secretion stimulated by KIC and KC without affecting the first one. It is plausible that the increased GABA release might restrict the availability of the amine to feed the "GABA-shunt" and this would in turn diminish the rate of ATP generation necessary to maintain sustained secretion. In support of this interpretation, it has recently been shown that islets of SUR1 knock-out mice which are permanently depolarized exhibit a diminished metabolic flow in the "GABA-shunt" [START_REF] Changhong | Elimination of K ATP channels in mouse islets results in elevated [U-13 C]glucose metabolism, glutaminolysis, and pyruvate cycling but a decreased γaminobutyric acid shunt[END_REF]. Therefore, it seems that GABA is the main metabolic fuel used during the stimulation of insulin secretion by BCKA. Another argument in support of this idea is that BCKA (KIC) block 2OGdh in the Krebs cycle favouring metabolism in the "GABA-shunt" (see diagram in Figure 6). In contrast, first phase release in response to KIC might be more dependent on a direct effect of the 2oxoacids on K + ATP channels at the sulfonylurea binding site that would close the channels irrespective of the ATP concentration [START_REF] Bränström | Direct inhibition of the pancreatic β-cell ATP-regulated potassium channel by αketoisocaproate[END_REF][START_REF] Heissig | Mechanism of the insulin-releasing action of α-ketoisocaproate and related αketo acid anions[END_REF].

At variance with KIC and KC, KMV-induced secretion was not significantly modified by K + -depolarization. The latter, as well as KIV, are predominantly transaminated by the cytosolic isoform of BCAT and the formed 2OG might be less efficiently transported into mitochondria to serve as a susbstrate of GABA-T (see diagram in Fig. 6). Both, KMV and KIV, may be substantially metabolized to succinic acid as reflected by the increased synthesis of Asp which probably arises from transamination of oxaloacetate. The stimulation of insulin secretion by KMV is perhaps more dependent on its partial catabolism in the citric acid cycle than either KIC or KC. eGABA did not improve KIC transamination or, as expected, KIC-induced insulin secretion. Neither did it rescue the stimulation of insulin secretion by KIC after depleting islet GABA content with 70 mM KCl. These data cast some doubt on the suitability of eGABA as a true analogue of GABA, a matter that is presently under investigation.

To sum up, BCKA stimulation of insulin secretion depends on their transamination rates with Glu that generates 2OG. As 2OGdh is inhibited by BCKA (KIC), 2OG is used preferentially by GABA transaminase resulting in an increased metabolic flux in the "GABA-shunt" and a decreased intracellular GABA level (Figure 6). The increased GABA metabolism would contribute to the maintenance of high ATP generation, as required for sustained insulin secretion. Groups of 40 islets each, pre-perifused without substrates for 45 min, were stimulated for 30 min (between vertical broken lines) with 10 mM of one out of four different branched-chain 2-oxocarboxylic acids (KIC, KMV, KIV and KC represent oxo-4methyloxopentanoate, oxo-3-methylpentanoate, oxo-3-methylbutyrate and oxohexanoate, respectively). Pre-perifusion conditions were then re-established during the last 25 min. Each perifusion was repeated a variable number of times (•, n=27 for KIC; ▲, n= 27 for KMV; □, n= 6 for KC; , n=6 for KIV). Symbols represent means ± S.E.M. Groups of 40 islets each, pre-perifused without substrates for 45 min, were stimulated for 30 min (between vertical broken lines) with 10 mM KMV alone (▲, n=27) or together with 5 mM gabapentin ( , n=6). Pre-perifusion conditions were then reestablished during the last 25 min. Symbols represent means ± S.E.M. Figure 3. Effect of ethyl-GABA (eGABA) on insulin secretion of perifused rat islets stimulated by either oxo-4-methylpentanoate (KIC) or oxo-3-methylpentanoate (KMV).

LEGENDS TO FIGURES

In Figure 3A, groups of 40 islets each, pre-perifused without substrates for 45 min, were stimulated for 30 min (between vertical broken lines) with 10 mM KIC alone (•, n=9) or together with 1 mM eGABA (○, n=9). In Figure 3B, islets were stimulated with 10 mM KMV alone (▲, n= 7) or together with 5 mM eGABA ( , n=7). Pre-perifusion conditions were then re-established during the last 25 min. Symbols represent means ± S.E.M. Groups of 40 islets each, pre-perifused without substrates for 45 min, were stimulated for 30 min (between vertical broken lines) with 10 mM KIC alone (•, n=27) or together with 70 mM KCl (○, n=27). Pre-perifusion conditions were then re-established during the last 25 min. Symbols represent means ± S.E.M. In Figure 5A, groups of 40 islets each, pre-perifused without substrates for 45 min, were stimulated for 30 min (between vertical broken lines) with 10 mM KMV alone (▲, n= 6) or together with 70 mM KCl ( , n=7). In Figure 3B, islets were stimulated with 10 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. TABLES Table 1. Effect of two inhibitors of branched-chain 2-oxoacids' transamination activity (gabapentin and 4-methylvaleric acid) on islet insulin secretion stimulated by several 2-oxo acids.

Three groups, each of ten islets, were incubated at 37 0 C for 1 hour in 1 ml of Krebs-Ringer bicarbonate buffered with 0.5 mM NaHCO 3 and 25 mM Hepes (pH=7.4) and supplemented with FFA-free BSA. The insulin accumulated in the incubation medium was radioimmunologically measured. (KIC, KMV and KIV are oxo-4-methyloxopentanoate, oxo-3-methylpentanoate and oxo-3-methylbutyrate, respectively; Gln means L-glutamine; n represents the number of experiments). Three groups each of 30 islets were pre-incubated for 1 hour with 10 mM Gln. After washing the islets twice with saline, in order to get free of Gln which would otherwise contaminate HPLC separation) they were further incubated for a second hour with 10 mM oxo-4methyloxopentanoate (KIC). α-Amino acids were quantified in islets and incubation medium by their emitted fluorescence after being derivatized with o-phftaldialdehyde and separated by reverse phase HPLC. ("n" represents the number of experiments).

IRI (pmol insulin / min x islet)

Biochemical

  Journal Immediate Publication. Published on 28 Jan 2009 as manuscript BJ20081731 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081731 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Experimental Materials Collagenase P was obtained from Roche Diagnostics S.L. (Barcelona, Spain). Bovine serum albumin (BSA), BCAT inhibitors, and most of the organic compounds were obtained from Sigma-Aldrich Química S.A. (Madrid, Spain). Rat insulin was from Linco Research, Inc. (St. Charles, Missouri, U.S.A.). Na 125 I was obtained from Amersham Iberica S.A. (Madrid, Spain). DL-α-mehylleucine was from Acros Organics (NJ, U.S.A.). Inorganic compounds and organic solvents were obtained from VWR International Eurolab S.L.(Spain).

Biochemical

  Journal Immediate Publication. Published on 28 Jan 2009 as manuscript BJ20081731 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081731

Biochemical

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  Biochemical Journal Immediate Publication. Published on 28 Jan 2009 as manuscript BJ20081731 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081731A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

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Biochemical

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  and cerebellum: it almost abolished the enzymatic activity in islet homogenates (results not shown).

Biochemical

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Figure 1 .

 1 Figure 1. Stimulation of insulin secretion in rat perifused islets by branched-chain 2oxocarboxylic acids.

Figure 2 .

 2 Figure 2. Effect of gabapentin, a specific inhibitor of the cytosolic form of the branched-chain 2-oxoacid transaminase, on insulin secretion of perifused islets stimulated by oxo-3-methylpentanoate (KMV).

Figure 4 .

 4 Figure 4. Effect of 70 mM KCl depolarization on insulin secretion of perifused rat islets stimulated by oxo-4-methylpentanoate (KIC).

Figure 5 .

 5 Figure 5. Effects of 70 mM KCl depolarization on insulin secretion of perifused rat islets stimulated by either oxo-3-methylpentanoate (KMV) or oxohexanoate (KC).

Biochemical

  Journal Immediate Publication. Published on 28 Jan 2009 as manuscript BJ20081731 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081731 A c c e p t e d M a n u s c r i p t mM KC alone (■, n=6) or together with 70 mM KCl (□, n=6). Pre-perifusion conditions were then re-established during the last 25 min. Symbols represent means ± S.E.M.

Figure 6 .

 6 Figure 6. Scheme of the metabolic interconnections between the citric acid cycle and the "GABA-shunt".

Table 3 .

 3 Evaluation of the rate of transamination of different branched-chain 2oxoacids in isolated islets (in vivo transamination rates) at different L-glutamine (Gln) concentrations.Three groups each of 30 islets were pre-incubated for 1 hour with the Gln concentration indicated. After washing the islets twice with saline, they were further incubated for a second hour in the presence of one of three different branched-chain 2-oxoacids (KIC, KMV and KIV representing oxo-4-methyloxopentanoate, oxo-3methylpentanoate and oxo-3-methylbutyrate, respectively) at 10 mM. α-Amino acids were measured in islets and incubation medium. The rate of transamination was calculated as the amount of branched-chain α-amino acid (Leu, Ileu and Val) released into the incubation medium. α-Amino acids were quantified by their emitted fluorescence after being derivatized with o-phftaldialdehyde and separated by reverse phase HPLC. ("n" represents the number of experiments).

FIGURESFigure 2 Biochemical

 2 FIGURES

  Figure 4

Figure 6

 6 Figure 5B

Table 2 .

 2 Effect of three enzyme inhibitors (gabapentin, 4-methylvaleric acid and DL-methylleucine) on branched-chain 2-oxoacids' transamination activity of islet homogenates (in vitro transamination rates). ) and L-glutamate (1 and 2 mM, respectively). The α-amino acids were quantified by their fluorescence after being derivatized with o-phftaldialdehyde and separated by reverse phase HPLC. ("n" represents the number of experiments).

	Isolated islets were sonicated in a lysis buffer defined in the experimental section. The enzymatic reaction
	was measured as the amount of branched-chain α-amino acid (Leu, Ileu, Val, nor-Leu) linearly
	accumulated at saturating concentrations of the corresponding 2-oxoacid (KIC, KMV, KIV and KC
	representing	oxo-4-methyloxopentanoate,	oxo-3-methylpentanoate,	oxo-3-methylbutyrate	and
	oxohexanoate, respectivelytransamination rate (pmol / islet x 20min)
	Gabapentin		KIC	n	p		KMV	n	p	KIV	n	p	KC	n	p
	-		24,27 ± 1,30	3	-	12,66 ± 0,97	7	-	26,20 ± 2,39 6	-	10,27 ± 0,15 3	-
	2 mM		23,35 ± 1,97	3	N.S		9,58 ± 0,64	7 <0,02	17,72 ± 1,91 6 <0,02	8,52 ± 1,58	3	N.S
	10 mM		19,04 ± 2,75	3	N.S		6,58 ± 0,71	3 <0.005 15,24 ± 1,18 3 <0.02	7,18 ± 1,21	3	N.S
	50 mM		9,51 ± 1,71	3 <0.008		2,51 ± 0,98	3 <0.001	4,15 ± 0,28	3 <0.001	4,56 ± 0,80	3 <0.002
	4-methylvaleric acid	KIC	n	p		KMV	n	p	KIV	n	p
	-		16,54 ± 0,38	2	-		15,53 ± 0,84 3	-	20,03 ± 2,05 3	-
	2 mM		7,88 ± 1,09	3 <0.009		7,73 ± 0,60	3 <0.002	9,80 ± 0,74	3 <0.009
	10 mM		1,99 ± 0,23	3 <0.001		3,91 ± 0,94	3 <0.001	5,05 ± 0,86	3 <0.002
	20 mM		0,56 ± 0,32	3 <0.001		1,76 ± 0,87	3 <0.001	2,59 ± 0,63	3 <0.001
	DL-methylleucine	KIC	n	p
	-		22,53 ± 1,92	3	-
	2 mM		18,96 ± 2,19	3	N.S
	10 mM		15,11 ± 1,64	3 <0.04
	20 mM		12,73 ± 2,53	3 <0.04

Table 4 .

 4 Effect of 70 mM KCl depolarization and/or 10 mM oxo-4methyloxopentanoate (KIC) on islet content and release of α-amino acids.

Table 5 .

 5 Concentration-dependent suppression of islet 2-oxoglutarete dehydrogenase activity by oxo-4-methylpentanoate (KIC). Isolated islets were sonicated in a lysis buffer defined in the experimental section. The enzymatic reaction was measured as the amount of NADH produced at saturating concentrations of 2oxoglutarate (5 mM) and NAD + (2.5 mM). ("n" represents the number of experiments). Biochemical Journal Immediate Publication. Published on 28 Jan 2009 as manuscript BJ20081731 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081731 A c c e p t e d M a n u s c r i p t

	Islet amino acid content (pmol / ng DNA)
		GABA	p	Taurine	p
	control	4,61 ± 0,69 (7)	-	1,42 ± 0,24 (7)	-
	10 mM KIC	1,85 ± 0,11 (6)	<0.004	1,47 ± 0,42 (8)	N.S
	70 mM KCl	2,25 ± 0,29 (6)	<0.01	0,51 ± 0,11 (7)	<0.005
	10 mM KIC+70 mM KCl	1.55 ± 0,14 (6)	<0.002	0,74 ± 0,08 (6)	<0.03
	Islet amino acid release (pmol / ng DNA)
		GABA	p	Taurine	p
	control	0,98 ± 0,29 (8)	-	0,31 ± 0,05 (6)	control
	10 mM KIC	1,02 ± 0,17 (6)	N.S	0,31 ± 0,05 (7)	N.S
	70 mM KCl	2,10 ± 0,37 (6)	<0.03	1,00 ± 0,27 (7)	<0.04
	10 mM KIC+70 mM KCl	2,38 ± 0,47 (6)	<0.02	0,81 ± 0,16 (7)	<0.02

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

' 2009 The Authors Journal compilation ' 2009 Portland Press Limited

ACKNOWLEDGMENTS

This work was partially supported by grant PI-06/1744, from Instituto de Salud Carlos III, Madrid (Spain). Inés Hernández-Fisac was a pre-doctoral fellow supported during four years by the Ministerio de Educación y Ciencia, Spain. Javier Pizarro Delgado is a pre-doctoral fellow partially supported by private funding (Astellas Pharma S.A., Madrid, Spain).


Rosa Zaragozá

Luis Torres

Concha García

Pilar Eroles

Fernando Corrales

Ana Bosch

Ana Lluch

Elena R García-Trevijano

Juan R Viña
email: juan.r.vina@uv.es

Nitration of cathepsin D enhances its proteolytic activity during mammary gland remodeling after lactation

Keywords: mammary gland involution, nitrotyrosine, post-translational modification, cathepsin D activity MMP, matrix metalloproteinase, ECM, extracellular matrix, iNOS, inducible nitric oxide synthase, ONOO -, peroxynitrite, CD, cathepsin D, WT, wild-type, Psp. A, pepstatin A, NO, nitric oxide

Proteomic studies in the mammary gland of control lactating and weaned rats have shown that there is an increased pattern of nitrated proteins during weaning when compared to controls. Here we report the novel finding that cathepsin D is nitrated during weaning. The expression and protein levels of this enzyme are increased after 8 hours of litter removal and this up-regulation declines 5 days after weaning. However, there is a marked delay in cathepsin D activity since it does not increase until 2 days postweaning and remains high thereafter. In order to find out whether nitration of cathepsin D regulates its activity, iNOS -/-mice were used. The expression and protein levels of this enzyme were similar to wild type animals but the proteolytic activity was significantly reduced during weaning in knock-out when compared to wild type mice. In vitro treatment of recombinant human cathepsin D or lactating mammary gland homogenates with relatively low concentrations of peroxynitrite enhances the nitration as well as specific activity of this enzyme. Using mass spectrometry it has been shown that residue Tyr 168 was nitrated. All these results show that protein nitration during weaning might be a signaling pathway involved in mammary gland remodeling.

removal [START_REF] Clarkson | NF-κB inhibits apoptosis in murine mammary epithelia[END_REF]. Downstream targets of this pathway are up-regulated, ensuring the transition to the second phase of involution. In previous studies, we have demonstrated an induction of iNOS gene expression (NOS-2, inducible nitric oxide synthase), regulated by NF-κB, during weaning, resulting in an increase in nitric oxide (NO) levels and, subsequently, increased protein nitration [START_REF] Zaragozá | Weaning induces NOS-2 expression through NF-kappa modulation in the lactating mammary gland: importance of GSH[END_REF].

Nitration is a post-translational modification of tyrosine residues of proteins mediated by peroxynitrite (ONOO -). Nitration of tyrosine or tyrosyl groups of a protein modulates protein function and is involved in signal transduction pathways, which lead to an alteration of cellular metabolism and functions. In particular, ONOO --induced protein oxidation often leads to a loss of protein activity [START_REF] Macmillan-Crow | Peroxynitrite-mediated inactivation of manganese superoxide dismutase involves nitration and oxidation of critical tyrosine residues[END_REF][START_REF] Görg | Reversible inhibition of mammalian glutamine synthetase by tyrosine nitration[END_REF], seldom to activation [START_REF] Ji | Activation of microsomal glutathione S-transferase by peroxynitrite[END_REF][START_REF] Ji | Nitration of tyrosine 92 mediates the activation of rat microsomal glutathione S-transferase by peroxynitrite[END_REF][START_REF] Rebrin | Nitration of tryptophan 372 in succinyl-CoA:3-ketoacid CoA transferase during aging in rat heart mitochondria[END_REF]. Because of its recognized significance, there is an increasing interest to identify nitrated proteins in order to gain a better understanding of their involvement in different biological processes, as is the case for mammary gland remodeling. In the present study, using mass-spectrometry-based proteomic technology, the pattern of nitrated proteins during weaning was analyzed and compared to control lactating mammary glands. We have identified cathepsin D (CD) (EC 3.4.23.5) within the nitrated proteins found after weaning, and for the first time we describe this post-translational modification on this enzyme.

CD is a major aspartic proteinase of endosomes and lysosomes and it is a member of the pepsin family of proteinases [START_REF] Tang | Evolution in the structure and function of aspartic proteases[END_REF]. It is involved in the turnover of cellular proteins as well as in the selective processing of MHC II antigens, hormones, and growth factors, in the range of pH 2.8-5.0. Gene knockout studies have demonstrated that cathepsin D-deficient mice die prematurely from massive destruction of lymphoid organs and progressive atrophy of the intestinal mucosa, suggesting essential functions of CD in tissue homeostasis [18]. CD is synthesized as an inactive 52 KDa pre-pro-enzyme that undergoes sequential proteolytic cleavages to produce the mature form. In lysosomes, the removal of 44 amino acids from the amino terminus yields an active 48 KDa single-chain molecule. This single chain form is then cleaved by auto-processing and, mainly, by cysteine proteinases into a mature two-chain enzyme consisting of a light 14 KDa amino-terminal domain and a heavy 34 KDa carboxylterminal domain [START_REF] Rochefort | Cathepsin D in cancer metastasis: a protease and a ligand[END_REF]. CD is increased during weaning, playing a key role in protein degradation during mammary gland apoptosis; indeed, CD is known to be involved in the mitochondrial caspase-dependent intrinsic pathway of cell death [20]. Despite the relevance of this proteinase in physiological and pathological events, few data exist on the factors that modulate its secretion, activity, and extracellular function in lactating mammary tissues.

Recently it has been demonstrated that prolactin controls the processing and polarized secretion of CD in this tissue [START_REF] Castino | Prolactin promotes the secretion of active cathepsin D at the basal side of rat mammary acini[END_REF]. On the other hand, interferon-γ activates Stat-1 resulting in decreased vacuolar pH which greatly affects CD processing and secretion [START_REF] Khalkhali-Ellis | IFN-γ regulation of vacuolar pH, cathepsin D processing and autophagy in mammary epithelial cells[END_REF]. In regard to CD activation, here we report the novel finding that CD is nitrated during weaning; Tyr 168 is identified as the modified residue. Moreover, it is noteworthy that in vivo and in vitro nitration of CD enhances its proteolytic activity. All these results highlight the importance of protein nitration as part of the physiological events that take place at the end of lactation, when the mammary gland is prepared for the next pregnancy/lactation cycle.

approved by the Council of The American Physiological Society. The Research Committee of the School of Medicine (University of Valencia, Valencia, Spain) approved the study protocol. Following parturition, the litters were maintained with at least 10 pups in rats and 7 pups in the case of mice.

At the peak of lactation (days 12-14), the rats were divided into different groups: Control lactating rats (n=8) or weaned rats from which pups were removed 12 days after delivery to initiate involution. Mammary tissue samples were collected at the indicated times after weaning (at least three rats for each condition were used). In the case of mice, the groups were as follows: Control mice at the peak of lactation (days 9-11) or weaned animals from which the litter was removed 6, 24, 48 and 72 hours before sacrifice.

The animals were anesthetized with sodium pentobarbital (60 mg/kg body wt. in 0.9% NaCl i.p.; Abbott Laboratories, S.A.) and killed immediately after removing the inguinal mammary glands that were snap frozen in liquid nitrogen and stored at -80ºC for further analysis.

Antibodies

Depending on the species and the forms of CD to be detected, different CD antibodies were used in the Western blots. A rabbit polyclonal anti-human CD antiserum (Santa Cruz Biotech. SC-10725, Santa Cruz, CA, USA) was used to determine the expression of pro-CD and mature single chain forms of CD in rats and mice. This antibody was also used for CD immunoprecipitation. The Santa Cruz antibody is made to an N-terminal peptide of the mature single chain and should thus be able to detect the pro form, the single chain form and the light chain of double chain form, which includes the N-terminus. Nonetheless, we could only detect the pro and the mature single chain forms. For this reason, other anti-CD antibodies were used: a rat monoclonal anti-mouse CD antiserum (Clone: 204712) from R&D Systems (Minneapolis, USA) was used in blots from mice homogenates to visualize the mature double chain forms of CD, both the heavy and light chains. Finally, a mouse monoclonal anti-human CD antibody (Clone: BC011) from Calbiochem (Schwalbach, Germany) was used to determine the expression of recombinant CD from human liver and in the immunoblots performed after immunoprecipitation of CD. To corroborate "in vitro" nitration with peroxynitrite, a mouse monoclonal NO-tyrosine antibody (Clone 1A6) was used (Upstate, Lake Placid, NY, USA).

Immunoblotting

Protein expression was evaluated by a standard immunoblotting procedure. Mammary gland tissues were homogenated in a buffer containing detergents and protease inhibitors as described previously [START_REF] Zaragozá | Inhibition of liver trans-sulphuration pathway by propargylglycine mimics gene expression changes found in the mammary gland of weaned lactating rats: role of glutathione[END_REF]. 20µg of whole extract proteins, quantified by the Bradford assay, were denatured with Laemmli sample buffer, separated by electrophoresis on a 12% polyacrylamide gel (Bio-Rad, Munich, Germany), and then electroblotted onto a nitrocellulose membrane (Protran®, Whatman, Kent, UK). After addition of the corresponding primary antibody, immunocomplexes were revealed by using a peroxidase-conjugated secondary antibody (Santa Cruz Biotech.), as appropriate, and subsequent peroxidase-induced chemiluminiscence reaction (ECL, Amersham Pharmacia Biotech., Buckinghamshire, UK).

Immunoprecipitation

Tissue lysates obtained from lactating and weaned mammary glands were precleared with protein A and G-Sepharose (50:50 v/v) (Amersham Pharmacia Biotech) for 1 h at 4ºC and centrifuged for 5 min. The supernatant was incubated first with the anti-cathepsin D antibody from Santa Cruz Biotech overnight at 4ºC, and then with protein A and G-Sepharose (50:50 v/v) for 4 h at 4ºC on a rotating device. Pellets were collected by centrifugation at 12000 rpm for 1 min, 4ºC. The supernatants were discarded, and each pellet was subsequently washed three times with Tris-HCl pH 8.0. After washing the sepharose, the immunocomplexes were eluted by boiling in the SDS-gel sample buffer for 5 min and then subjected to SDS-PAGE immunoblot as described previously. Representative results of three separate experiments are shown.

Pepstatin A-affinity purification of CD Homogenates from weaned mammary gland (500 µg of whole extract proteins) were incubated for 15h at 4ºC with pepstatin A-agarose coated beads (Sigma) in 20mM citrate phosphate buffer pH 7.0, according to Follo et al. [START_REF] Follo | Folding, activity and targeting of mutated human cathepsin D that cannot be processed into the double-chain form[END_REF].Then, the mixture was centrifuged at 20000g for 5 min. and the pellet was washed with 20mM citrate phosphate buffer pH 7.0. Finally the pellet was re-suspended and denatured in 30µl of sample loading buffer prior to western blotting analysis.

Two-Dimensional Electrophoresis

Mammary gland tissue from each condition was homogenized in rehidratation buffer from BioRad (8M Urea, 2% CHAPS, 50mM DTT, 0.2% Bio-Lyte ® 3/10 ampholyte, 0.001% Bromophenol blue). After cleaning up the samples with ReadyPrep 2-D Cleanup kit (BioRad), 500 µg of tissue lysates were loaded on precast immobilized pH gradients (IPG) gel strips pH 5-8 (BioRad) and subjected to IEF two-dimensional (2D) PAGE. The isoelectrofocusing conditions were those recommended by the manufacturer for the type of strip used. Second dimension gels contained 8% acrylamide. Simultaneously, proteins were resolved by 2D PAGE and then electroforetically transferred to nitrocellulose membranes (Protran®, Whatman) or visualized using silver or coomassie stainings. Membranes were probed with a 1:1000 dilution of anti-nitrotyrosine antibody (Upstate) as described above. Spots corresponding to nitrated proteins were excised manually from coomassie-stained gels and identified by mass spectrometry.

Protein identification by Mass Spectrometry

Protein preparation and tryptic digestion. Protein samples corresponding to weaned mammary gland lysates recovered from 2D gels, were precipitated with 20% TCA on ice and, after centrifugation, the pellet was washed with cold acetone. The same procedure was performed in the case of human CD (25 µg) treated with 20 µM peroxynitrite or with 1.2 N NaOH , in order to identify the nitration site of the enzyme. Precipitated proteins were resuspended in 25 mM ammonium bicarbonate and digested with 12.5 ng/µl trypsin for 12 h at 37º C. LC-ESI-MS/MS analysis. MS/MS analysis was performed as previously described [START_REF] Muñoz | Mass spectrometric characterization of mitochondrial complex I NDUFA10 variants[END_REF]. Microcapillary reversed phase LC was performed with a CapLC TM (Waters) capillary system. Reversed phase separation of tryptic digests was performed with an Atlantis, C 18 , 3 µm, 75 µm x 10 cm Nano Ease TM fused silica capillary column (Waters) equilibrated in 5% acetonitrile, 0.2% formic acid. After injection of 6 µl of sample, the column was washed for 5 min with the same buffer and the peptides were eluted using a linear gradient of 5-50% acetonitrile in 45 min at a constant flow rate of 0.2 µl/min. The column was coupled online to a Q-TOF Micro (Waters) using a PicoTip nanospray ionization source (Waters). The heated capillary temperature was 80º C and the spray voltage was 1.8-2.2 kV. MS/MS data were collected in an automated data-dependent mode. The three most intense ions in each survey scan were sequentially fragmented by collision-induced dissociation (CID) using an isolation width of 2.0 and a relative collision energy of 35 V. Data processing was performed with MassLynx 4.1. Database searching was carried out with ProteinLynx Global Server 2.1 (Waters) and Phenyx 2.2 (GeneBio, Geneva, Switzerland) against Uniprot knowledgebase Release 12.3 consisting of UniprotKB/Swiss-Prot Release 54.3 and UniprotKB/TrEMBL Release 37.3 with 285.335 and 4.932.421 entries, respectively. The search was enzymatically constrained for trypsin and allowed for one missed cleavage site. Further search parameters were as follows: no restriction on molecular weight and isoelectric point; fixed modification, carbamidomethylation of cysteine; variable modification, oxidation of methionine. Data were then manually inspected and the results were only accepted when the Mr and pI of the identified protein were coincident with the electrophoretic mobility of the corresponding spot on 2D.

RNA Isolation and quantitative RT-PCR RNA was purified using TriZol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA (5ng) was reverse transcribed and the cDNA products amplified by qPCR using the GeneAmp Fast PCR Master Mix (Applied Biosystems, Foster City, CA, USA). All reactions were carried out in triplicate. Quantitative real-time PCR was run in the 7900HT Fast Real-Time PCR System. Pre-developed Taqman primers specific for CD and 18S were purchased from Applied Biosystems. Results were normalized according to 18S quantification in the same sample reaction. Gene expression values were achieved by employing the equation:

Relative amount = 2 -∆Ct wherein ∆Ct stands for the difference between Ct values from the gene of interest and 18S.

Enzymatic assay of Cathepsin D

Cathepsin D protease activity was measured in the samples using the CD assay kit from Sigma (St. Louis, MO, USA) based on the method of Yasuda et al. [START_REF] Yasuda | Characterization of new fluorogenic substrates for the rapid and sensitive assay of cathepsin E and cathepsin D[END_REF]. Briefly, 5 µl of tissue homogenates (1mg protein/ml) from lactating rats and mice or human CD (2.4 µg/ml) (Sigma) were added to the assay buffer pH 3.5, followed by the addition of the quenched fluorometric substrate 7-methoxycoumarin-4-acetic acid at a final concentration of 20µM. The fluorescence released by the action of the enzyme was measured at 37ºC in a fluorometric plate reader during 60 minutes incubation. To prove that substrate was hydrolyzed by CD only, the specific inhibitor pepstatin A (Psp. A) from Sigma, was added to parallel samples at a final concentration of 0.2 mg/ml to inhibit the CD activity (data not shown). Samples were assayed at least three times in triplicate. Data are given as the mean value ±SEM.

In vitro protein nitration

Peroxynitrite was purchased from Calbiochem, suspended in 1,2N NaOH and stored in an oxygen-free atmosphere at -80ºC until use. Prior to experimentation, the concentration of ONOO -was measured by the increase in absorbance at 302nm (ε 302nm = 1,670 M -1 ⋅cm -1 ) in 1.2N NaOH [START_REF] Radi | Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide[END_REF]. 20µg of recombinant CD from human liver (Sigma) or 50µg of proteins from control lactating mammary gland homogenates were reacted with 0-500µM ONOO -in assay buffer, pH 3.5 (from the CD assay kit) in the presence or absence of 30µM (-) epicatechin (Sigma) in a total volume of 50µl. The reaction was performed by placing a small aliquot of ONOO -in a tube containing the sample, immediately followed by 20 min incubation. In control tubes, the samples were treated with equal volumes of 1.2N NaOH (vehicle). Aliquots were taken for Western blot analysis and determination of enzymatic activity.

Statistics

Data are reported as means ±SEM. In figures 4A and 5A, Student's t test with the Bonferroni correction was used for comparison between two groups. This analysis was also used in figures 3A and B to compare WT and KO mice at the same points of weaning. A difference was considered to be statistically significant when p < 0.05. For the rest of the figures, statistical significance was evaluated using one-way ANOVA. The homogeneity of the variances was analyzed by the Levene test; in those cases in which the variances were unequal, the data were adequately transformed before ANOVA. The null hypothesis was accepted for all the values of these sets in which the F-value was non-significant at p>0.05. The data for which the F-value was significant were examined by Tukey's test at p<0.05.

RESULTS AND DISCUSSION

Nitroproteomic analysis of lactating and weaned mammary gland from rats

In our previous work, using chromatin immunoprecipitation assays, it was demonstrated NF-κB binding to the iNOS promoter at the early onset of events triggered during weaning, resulting in the current transcription of the gene. This induction resulted in overproduction of NO during weaning, which led to an increase in the protein nitration pattern; however, this post-translational modification was limited to a few specific proteins [START_REF] Zaragozá | Weaning induces NOS-2 expression through NF-kappa modulation in the lactating mammary gland: importance of GSH[END_REF].

The question raised in this study was to elucidate the functional consequences of protein nitration in our physiological model. Previous reports indicate that the nitration of protein tyrosine residues can alter protein function, either decreasing [START_REF] Macmillan-Crow | Peroxynitrite-mediated inactivation of manganese superoxide dismutase involves nitration and oxidation of critical tyrosine residues[END_REF][START_REF] Görg | Reversible inhibition of mammalian glutamine synthetase by tyrosine nitration[END_REF] or increasing [START_REF] Ji | Activation of microsomal glutathione S-transferase by peroxynitrite[END_REF][START_REF] Ji | Nitration of tyrosine 92 mediates the activation of rat microsomal glutathione S-transferase by peroxynitrite[END_REF][START_REF] Rebrin | Nitration of tryptophan 372 in succinyl-CoA:3-ketoacid CoA transferase during aging in rat heart mitochondria[END_REF] the catalytic activity. To identify the nitrated proteins in the weaned mammary gland, proteomic studies were performed. Figure 1 shows representative silver stained gels (Panel A) and nitrated proteins (Panel B) of whole tissue extracts from lactating and 72 h weaned mammary glands. As seen in figure 1B, protein nitration is increased during weaning. Nitrated proteins were identified using a combination of procedures that included 2D-PAGE immunoblotting with specific antibodies against nitrotyrosine residues and mass spectrometry of proteins recovered from 2D gels. Twenty proteins in the molecular mass range of 15 to 75 kDa and with an isoelectric point between 5 and 8 were identified by 2D IEF and MALDITOF-MS. Since milk stasis plays an important role in the process of involution of the mammary gland after weaning, we were interested in the complete protein profile after weaning and therefore we did not use milk-depleted mammary tissue for our proteomic analysis. In consequence, we were unable to detect low abundant proteins, and therefore we cannot rule out the possibility that more proteins could be nitrated after weaning. The most general conclusion from the analysis of the protein map shown is a relative abundance of albumin, immunoglobulin chains, and casein fragments (data not shown), which is not surprising, due to milk stasis in the weaned mammary gland. Nevertheless, it is possible that these fragments of nitrated-albumin could play a more important role than one might think at first. Indeed, it has been described that the injection of small peptides derived from milk into the mammary gland accelerates the process of involution [START_REF] Shamay | Casein-derived phosphopeptides disrupt tight junction integrity, and precipitously dry up milk secretion in goats[END_REF]. However, a 48 KDa nitrated protein was identified, with coverage of 2.5%, as cathepsin D in mammary gland 72 h after weaning.

An immunoprecipitation with CD antibody, followed by immunoblotting against nitrotyrosine residues, further confirmed the nitration of this enzyme after 72 h weaning. Two different forms of CD immunoprecipitated from the lactating mammary gland homogenates were nitrated. One is the mature single chain (msc) CD above the 43 KDa molecular weight markers. The other corresponds to the heavy chain (34 KDa) from the mature double chain (mdc) form (Fig 1C left panel). Since it has been described that the msc accumulates in endosomes whilst the mdc is mainly found in lysosomes [START_REF] Castino | Prolactin promotes the secretion of active cathepsin D at the basal side of rat mammary acini[END_REF], the results presented here suggest that nitration takes place along the endosomal-lysosomal compartment. In the right panel, equal loading of both samples was assessed by loading on the gel 1% of control or weaned mammary gland homogenate (Input). Besides, to corroborate that CD was immunoprecipited, a western blot against CD was performed in the immunoprecipitate from 72h weaned mammary gland (Fig. 1C right panel).

Since the relevance of cathepsin D nitration was critical to our findings, we wanted to ensure that this post-translational modification took place in our experimental model. Thus, another approach to purify CD from our samples was used. Pepstatin A (Psp A) is an inhibitor of CD that fits into the active site of the enzyme; we used Pst A-coated agarose beads to efficiently precipitate CD from weaned mammary gland homogenates. Those beads were incubated with 72h weaned mammary gland homogenates, extensively washed and the bound fractions were analysed by western blotting, as described in the Experimental section. The detection of CD is shown in figure 1D and, the same membrane was reprobed with antinitrotyrosine antibody, which also immunoreacted with the same bands. This fact further demonstrated that CD becomes nitrated during weaning.

Effect of weaning on the expression and activity of cathepsin D in rat lactating mammary gland

Since CD proteolytic activity has been generally involved in the apoptotic process, and forced weaning is known to induce apoptosis in lactating mammary tissue [START_REF] Green | Apoptosis regulation in the mammary gland[END_REF], CD mRNA and protein levels were studied at different times of weaning. Western blot analysis of lactating and weaned mammary gland lysates revealed two major CD-related bands that correspond to the enzymatically inactive precursor of 52 KDa and the mature single chain form of 48 KDa. Figure 2A shows that the amount of the latter (msc) is increased after 8 h of pup removal and remains elevated 3 days later, being barely detectable thereafter. This increase in protein levels correlates with the accumulation of CD mRNA, measured by real time RT-PCR, which is maximal at 8 and 24 h after weaning (Fig 2B).

An enhancement of enzyme activity was expected due to the fact that the amount of msc CD increased with weaning (Fig 2A). However, when measuring CD proteolytic activity, it is noteworthy that it was not increased until 48 h after pup removal (Fig 2C). Although previous authors have reported a slight decrease in the proteolytic activity of this enzyme in rats weaned for 24 h [START_REF] Hernández-Montes | Changes in cathepsin D activity of maternal tissues during lactation and weaning in rats[END_REF], this was followed by an additional increase with maximal activity 4 days later [30]. This delay in the activation of the enzyme when compared to its protein levels suggests that a post-translational modification of CD might be regulating its activity in our experimental model. In fact, we have described the physiological nitration of this proteinase during weaning (Fig 1C), and we hypothesize that this nitration could be involved in the modulation of CD activity.

Evaluation of expression and activity of cathepsin D in iNOS -/-and WT mice

In order to study the possible role of nitric oxide in the regulation of CD activity, studies were performed using knock-out mice for the iNOS gene. Those mice have an apparently normal lactation cycle and no clear differences were seen between iNOS -/-and wild type mice with respect to milk production and histological structures of the mammary gland (data not shown). As seen in Fig 3A, the activity of CD in weaned-glands of WT and KO mice was increased after 2 and 3 days post-weaning. Interestingly, when measuring CD activity, we found that mice deficient for iNOS showed decreased protease activity throughout involution compared to wild type animals (Fig 3A).

Since nitric oxide (NO) could be involved in the regulation of CD expression, we analyzed gene expression levels of this enzyme by real time PCR. However, when analyzing CD mRNA expression, no statistical differences were found between WT and iNOS -/-mice at each experimental point studied (Fig 3B). Furthermore, the increased pattern of expression seen in rats (Fig 2B) was similar to that observed in both types of mice, in which CD expression was increased at 1, 2 and 3 days post-weaning (Fig 3B).

On the other hand, NO could also be involved in protein stability or translational regulation, therefore CD protein levels were studied in WT and iNOS -/-mice. Western blot analysis demonstrated that there was no change in the protein levels of CD in KO versus WT mice at the same experimental time points. In both types of mice, the protein levels increased during weaning in a time-dependent manner. This increase was maximum for the pro-CD ( 52kDa) 2 days after weaning. The mature single chain (CD msc) was cleaved yielding the mature double chain form of CD (heavy and light chains were detected by the antibody giving immunoreactive bands of 30 and 14 kDa, respectively). In conclusion, the results obtained with iNOS -/-mice emphasize the importance of NO in CD activity regulation.

Effect of in vitro peroxynitrite-induced nitration on cathepsin D activity: Studies in rat mammary gland homogenates and in human recombinant cathepsin D.

Mammary gland homogenates from control lactating rats were incubated with 0 to 500 µM peroxynitrite (ONOO -). CD activity was 50% higher at 20µM ONOO -, compared to the control, but this increase was abolished in the presence of (-) epicatechin (Fig 4A). The flavonoid epicatechin has been shown to interfere with protein tyrosine nitration by intercepting the tyrosil radical [START_REF] Schroeder | Epicatechin prevents nitration but not oxidation reactions of peroxynitrite[END_REF]. However, at concentrations higher than 50µM ONOO - there was a linear decrease in activity (data not shown). This observation can be attributed to the simultaneous oxidative effects of ONOO -on a variety of amino acid residues in addition to nitration of aromatic amino acids [START_REF] Alvarez | Peroxynitrite reactivity with amino acids and proteins[END_REF]. Since we wanted to study nitration within a physiological range, further characterization of the effects of high concentrations of ONOO - was not pursued.

Western blot with 3-nitrotyrosine antibodies showed that protein nitration was enhanced after the incubation with 20µM ONOO -and was undetectable either in control lactating rats treated with vehicle or with ONOO -and epicatechin, a nitration inhibitor (Fig 4B ). In order to assess whether CD was nitrated after the incubation with 20µM ONOO -, immunoprecipitation of control and nitrated samples with CD antibody was performed (Fig 4C ). CD was immunoprecipitated from tissue lysates incubated with vehicle or ONOO -, and analyzed for tyrosine nitration by Western blotting. The results showed that nitrated CD was increased after peroxynitrite-induced nitration (Fig 4C right panel).

Although we have described for the first time CD nitration and this post-translational modification seems to correlate with increased catalytic activity, other proteins, also affected by NO, could be modulating CD activity indirectly. To gain a better understanding of the mechanisms by which protein nitration might affect CD activity, in vitro experiments with recombinant CD from human liver were performed. As seen in figure 5A, the presence of peroxynitrite produced an increase in human CD activity. Epicatechin (30µM) completely abolished tyrosine nitration of recombinant human CD by 20µM peroxynitrite (Fig 5B). Moreover, epicatechin completely prevented the induction of CD activity by peroxynitrite (Fig. 5A), corroborating that this enzymatic activity is specifically modulated by tyrosine nitration.

The next question raised by this study is how nitration of this amino acid can affect CD activity. As already mentioned, immature CD can be partially autoactivated leading to the mature form of the enzyme, therefore a specific nitration could be somehow accelerating or interfering with the autoprocessing of the immature form of CD. However, our experiments with wild type and knock-out animals show the same pattern of proteolytic CD cleavage (Fig 4C). Moreover, it has been shown that pro-CD requires lysosomal cysteine proteinases for the final processing of its propart and we have shown that recombinant human CD undergoes in vitro nitration and activation in a cysteine protease-free buffer. Therefore, we can exclude the autoprocessing as the mechanism for the observed nitration-induced activity of CD.

These data suggest a conserved nitration domain among species since both rat and human CD can be nitrated in vitro by peroxynitrite (Fig 4C and5B). Moreover, the fact that this nitration induced an increase in enzyme activity versus non-nitrated CD suggests that nitric oxide can modulate CD activity in a direct manner ( only in recombinant human CD, but also in tissue homogenates, suggesting that direct nitration of this enzyme is responsible for the enhanced activity observed.

Identification of the nitration site

It has been shown that tyrosine nitration can dramatically induce profound changes in protein structure and function due to a shift in the pKa of the tyrosine hydroxyl group [START_REF] Sokolovsky | Conversion of 3-nitrotyrosine to 3aminotyrosine in peptides and proteins[END_REF]. In order to identify the Tyr residue targeted by peroxynitrite, tryptic digests of nitrated and nonnitrated human recombinant CD were characterized by mass spectrometry. The doubly charged ion with m/z 823.92 was assigned as the nitrated form of peptide (159)LVDQNIFSFYLSR(171). All y ions from y4 showed the extra 45 Da suggesting the nitration of Tyr 168 . In contrast, no nitrations were detected when tryptic digests from control CD were analyzed. In particular, a doubly charged ion with m/z 801.42 was assigned as the non-nitrated (159)LVDQNIFSFYLSR( 171) peptide (Fig 6).

Nevertheless, although we could sequence most of the tyrosine residues present in the protein (13 Tyr residues out of 19), we were unable to sequence more than 40% of the human CD and therefore we cannot rule out the possibility that other tyrosines could also be nitrated. As shown in figure 7, Tyr 168 is found in the heavy chain of CD and is highly conserved among species and other proteases, such as human renin, porcine pepsin, and bovine chymosin. In fact, the detailed protein sequence analysis reveals a possible nitration domain with the sequence F S F/V Y X X R/S. Further studies would be needed in order to determine whether other proteinases with this conserved domain can also undertake an in vivo and in vitro nitration.

The important role played by pH in the modulation of CD activity has been extensively documented [START_REF] Khalkhali-Ellis | IFN-γ regulation of vacuolar pH, cathepsin D processing and autophagy in mammary epithelial cells[END_REF]34,[START_REF] Beaujouin | Cathepsin D overexpressed by cancer cells can enhance apoptosis-dependent chemo-sensitivity independently of its catalytic activity[END_REF]. Indeed, while an acidic pH lead to a fully active enzyme, at pH 7.5 a molecular bond between specific tyrosines and the catalytic aspartates stabilizes the protein structure in an inactive conformation [START_REF] Mása | Cathepsin D propeptide: Mechanism and regulation of its interaction with the catalytic core[END_REF]. Although Tyr 168 is 17.76 Å away from the active site and such a distance is not compatible with a direct hydrogen bond with the catalytic residues, it would be conceivable that the nitration of Tyr 168 could favor an irreversible active conformation of the enzyme by blocking a molecular bond crucial for CD folding. Moreover, an inhibitory domain has been localized at the N-terminus of CD [START_REF] Mása | Cathepsin D propeptide: Mechanism and regulation of its interaction with the catalytic core[END_REF]; at pH 7.5 the mature Nt is repositioned into the active site cleft, interacting with the catalytic residues. Since we cannot exclude the possibility of nitration in other non-identified tyrosine residues, the in vivo nitration of a specific Tyr at this site could block the binding of the inhibitory propeptide and allow the free access of the substrate.

Physiological and medical implications

All in all, in the future it would be very interesting to further investigate the mechanisms of modulation of CD activity induced by both nitration and nitrosylation, as well as the possibility of finding more tyrosine and/ or cysteine residues modified by nitric oxide. The relevance of these findings is granted by the fact that CD is known to be involved in pathological processes, such as inflammation, tumor progression, and metastasis. Indeed, CD is an independent marker of poor prognosis in human breast cancer [37,[START_REF] Benes | Cathepsin D-Many functions of one aspartic protease[END_REF]. Recently published studies have demonstrated a direct relationship between thrombin and CD up-regulation which would enhance angiogenesis, tumor growth and metastasis [START_REF] Hu | Thrombin up-regulates cathepsin D which enhances angiogenesis, growth and metastasis[END_REF]. Regarding to CD activity, is 8 to 16 times higher in the cytosol of mammary cancer cells than in normal cells [START_REF] Capony | Increased secretion, altered processing and glycosylation of pro-cathepsin D in human mammary cancer cells[END_REF]. A direct relationship between CD activity and the metastatic potential of tumors has been demonstrated [START_REF] Pujol | A prospective study of the prognostic value of cathepsin D levels in breast cancer cytosol[END_REF] although a mutated CD devoid of its catalytic activity also stimulates the growth of cancer cells, suggesting a novel mechanism by which CD could act as a mitogen in tumor progression [START_REF] Glondu | A mutated cathepsin-D devoid of its catalytic activity stimulates the growth of cancer cells[END_REF]. Nevertheless, little is known about the molecular mechanisms involved in the modulation of CD activity in all these processes, or how tyrosine-nitration could affect protein function. In summary, mammary gland involution is physiologic, orderly, and developmentally programmed. Thus, the involuting mammary gland is an ideal physiological model to search for genes involved in growth arrest, differentiation, and apoptosis. These processes are known to play an important role in cancer and other pathologies, therefore those genes relevant to involution in the normal mammary gland may be highly relevant in breast cancer. A pilot study using cDNA microarrays has demonstrated that through the analysis of differentially expressed genes during early mammary gland involution it may be possible to identify "novel" genes relevant to human breast cancer [START_REF] Blanchard | Gene expression profiling of early involuting mammary gland reveals novel genes potentially relevant to human breast cancer[END_REF]. This can also be applied to CD; the activation of this protein plays a key role in mammary gland involution, but more importantly, the involuting mammary gland offers an excellent experimental model for studying the mechanisms of CD activation that could also apply for breast cancer development. (A) Proteins from mammary gland homogenates were analyzed by SDS-PAGE and treated for immunoblotting as described in Materials and Methods. Two immunoreactive bands, corresponding to pro-cathepsin D (proCD) and mature single chain CD (CD msc) forms of CD were visualized in control and weaned rats using the CD antibody from Santa Cruz biotech. Position of the molecular weight markers (kilodaltons) is on the right. (B) CD mRNA expression in rat mammary tissue at the peak of lactation (control) and at different times of weaning, measured by real time RT-PCR. 18S was used as internal control (see Experimental section). Results are mean ± SEM for three independent experiments. (C) CD activity in mammary gland from control lactating and weaned rats by measurement of the hydrolysis of the fluorogenic substrate 7-methoxycoumarin-4-acetic acid as described in Materials and Methods (n= 4 per group). In (B) and (C), ANOVA was performed for the statistical analysis where different superscript letters indicate significant differences, P< 0.05; the letter "a" always represents the lowest value within the group. FIGURE 3. Expression of CD and proteolytic activity in iNOS -/-mice. (A) Lactating and weaned mammary glands were collected from wild type (WT) and inducible NOS null mice (KO) and analyzed for CD activity (n= 4 per group). (B) Gene expression of CD from wild type and iNOS -/-mice during lactation and weaning (n= 4 per group). In (A) and (B), to compare between the different conditions in WT or KO mice, ANOVA was performed for the statistical analysis where different superscript letters indicate significant differences, P< 0.05; the letter "a" always represents the lowest value within the group. Student's t test with the Bonferroni correction was used for comparison between WT and KO at each experimental time point, *, P<0.05. (C) Western blot analysis of CD at the peak of lactation (control) and during mammary gland involution (weaning) in wild type and iNOS -/-mice. The blot shows the presence of precursor (pro-CD) and mature single chain (CD msc) forms of CD recognized with the CD antibody from Santa Cruz Biotech. Since this antibody doesn't immunoreact with the mature double chain, the two degradation products of ∼30 KDa (CD heavy chain) and ∼14 KDa (CD light chain) are recognized with the CD antibody from R&D Systems. The blot shows a representative example from one individual mouse from each group. The molecular forms of CD are indicated on the left and the molecular weight markers (KDa) in the middle of both blots.

Biochemical

  Journal Immediate Publication. Published on 06 Jan 2009 as manuscript BJ20081746 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081746 A c c e p t e d M a n u s c r i p t

Biochemical

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  Fig 5A). One could argue that the protein context surrounding CD or the conformation of CD in vivo could be other than that favoring the nitration. Nevertheless, we have shown that CD from extracts of control lactating mammary gland can also be nitrated in vitro (Fig 4), as demonstrated for the recombinant protein, and that this nitration induces an increase in the enzymatic activity (Fig 4A). On the whole, our experiments show that a low degree of nitration increases CD catalytic activity, not Biochemical Journal Immediate Publication. Published on 06 Jan 2009 as manuscript BJ20081746 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081746A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 06 Jan 2009 as manuscript BJ20081746 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081746 A c c e p t e d M a n u s c r i p t

FIGURE 1 .

 1 FIGURE 1.Cathepsin D nitration in mammary glands from weaned rats. Protein expression profiles of control lactating and 72 hours weaned mammary glands separated by IEF 2D PAGE and silver stained (A) or staining of nitrated proteins by immunoblotting with anti-nitrotyrosine antibody (B). The pH increases from left to right on the abscissa and the molecular mass decreases from top to bottom on the ordinate. Molecular weight markers (kilodaltons) are on the left of the gel. (C) Tissue homogenates from control lactating or 72 h weaned (72 h W) mammary glands were immunoprecipitated (IP) by anti-cathepsin D antibody (Santa Cruz Biotech.), and the precipitates were then immunoblotted (IB) with antinitrotyrosine antibody (Upstate) or cathepsin D antibody (Calbiochem). Position of the molecular weight markers (kilodaltons) is on the left. (D) Mammary gland homogenates from 72h weaned rats (72h W) were incubated with pepstatin A-coated agarose beads. After elution, the bound fraction was analysed by western blotting, including an equal amount of starting homogenate (named INPUT). The membrane was immunoblotted (IB) with CD antibody (Calbiochem) and then stripped and re-probed with anti-NO tyrosine to verify protein nitration.

FIGURE 2 .

 2 FIGURE 2.Characterization of mammary gland CD from control lactating and weaned rats. (A) Proteins from mammary gland homogenates were analyzed by SDS-PAGE and treated for immunoblotting as described in Materials and Methods. Two immunoreactive bands, corresponding to pro-cathepsin D (proCD) and mature single chain CD (CD msc) forms of CD were visualized in control and weaned rats using the CD antibody from Santa Cruz biotech. Position of the molecular weight markers (kilodaltons) is on the right. (B) CD mRNA expression in rat mammary tissue at the peak of lactation (control) and at different times of weaning, measured by real time RT-PCR. 18S was used as internal control (see Experimental section). Results are mean ± SEM for three independent experiments. (C) CD activity in mammary gland from control lactating and weaned rats by measurement of the hydrolysis of the fluorogenic substrate 7-methoxycoumarin-4-acetic acid as described in Materials and Methods (n= 4 per group). In (B) and (C), ANOVA was performed for the statistical analysis where different superscript letters indicate significant differences, P< 0.05; the letter "a" always represents the lowest value within the group.

Biochemical

  Journal Immediate Publication. Published on 06 Jan 2009 as manuscript BJ20081746 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081746A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

FIGURE 4 .

 4 FIGURE 4. In vitro nitration of lactating mammary gland homogenates. Lactating mammary gland lysates were incubated with 0 or 20µM ONOO -(PN) in the presence or absence of (-) epicatechin. (A) The graph shows the increase in CD activity in the nitrated sample (20µM PN) when compared to control (treated with vehicle) or to lactating mammary gland homogenate treated simultaneously with PN and epicatechin (PN + Epic.). The increase in CD activity induced by PN was statistically significant. *, P<0.05. (B) A representative Western blot against tyrosine-nitrated residues or CD (with the Santa Cruz antibody) in samples from mammary tissue after treatment with ONOO -is shown. (C) Tissue homogenates from control lactating mammary glands treated with vehicle (control) or with ONOO -(20µM PN) were immunoprecipitated (IP) by anti-cathepsin D antibody (Santa Cruz Biotech.), and the precipitates were then immunoblotted (IB) with anti-nitrotyrosine antibody (Right panel) or cathepsin D antibody from Calbiochem (Left panel). Molecular weight markers (KDa) are indicated on each blot.

FIGURE 5 .

 5 FIGURE 5. Peroxynitrite-induced activation of cathepsin D. Commercial human CD reacted with 0 or 20µM ONOO -(PN) in the presence or absence of (-) epicatechin. This recombinant CD from human liver corresponds mainly to the heavy chain of the mature CD form (34 KDa). Samples were analyzed for tyrosine nitration or CD activity. (A) Effect of 20µM ONOO -on human CD activity. A significant increase (*, P<0.05) is observed after the addition of PN. (B) CD samples from human liver, treated with vehicle (control), ONOO -(PN) and ONOO -and epicatechin (PN + epic.), were analyzed for tyrosine-nitrated and overall CD (using the mouse monoclonal anti-human CD antibody from Calbiochem) by Western blot. Immunoblots are representative of three separate experiments and position of the molecular markers (KDa) is on the right.

FIGURE 6 .FIGURE 7 .

 67 FIGURE 6. Identification of the modified amino acid by mass spectrometry. Representative MS/MS spectra of tryptic digests from human cathepsin D (A) vehicle or (B) ONOO --treated are shown. The mass/charge value of 823.92 for the peptide (159)LVDQNIFSFYLSR(171) from CD treated with ONOO -contains a +45 Da adduct when compared to the vehicle-treated enzyme (m/z of 801.42), suggesting the nitration of Tyr 168 .

  Figure 1.

Figure 2

 2 Figure 2.

Figure 3

 3 Figure 3.

Figure 4

 4 Figure 4.

  Figure 5.

  Publication. Published on 06 Jan 2009 as manuscript BJ20081746 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081746 A c c e p t e d M a n u s c r i p t

Figure 6

 6 Figure 6.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Biochemical Journal Immediate Publication. Published on 06 Jan 2009 as manuscript BJ20081746 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081746 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGEMENTS AND FUNDINGS

We thank Mrs. Marilyn R. Noyes for kindly revising the manuscript. This work was supported in part by grants from Ministerio de Educación y Ciencia [BFU2007-62036 to J.R.V.; CIT-010000-2007-51 to L.T. and J.R.V. and Programa Ramón y Cajal to E.R.G-T]; Ministerio de Sanidad [FIS PI05/1332 to E.R.G-T] and Consellería de Sanidad [AP-061/08 to R.Z.]. R.Z is a fellow from Ministerio de Educación y Ciencia (Programa Juan de la Cierva).