Tyrosine nitration of carnitine palmitoyl transferase I during endotoxaemia in suckling rats

Biochimica et Biophysica Acta 1683 (2004) 1 – 6 www.bba-direct.com

Tyrosine nitration of carnitine palmitoyl transferase I during endotoxaemia in suckling rats Koji Fukumoto a, Agostino Pierro a, Victor A. Zammit b, Lewis Spitz a, Simon Eaton a,* a

Department of Paediatric Surgery, Institute of Child Health, 30, Guilford Street, London WC1N 1EH, UK b Hannah Research Institute, Hannah Research Park, Mauchline Road, Ayr, KA6 5HL, UK Received 17 December 2003; received in revised form 5 March 2004; accepted 31 March 2004 Available online 20 April 2004

Abstract Heart carnitine palmitoyl transferase I (CPTI) is inhibited in vivo during endotoxaemia and in vitro by peroxynitrite but the biochemical basis of this inhibition is not known. The aim of this study was to determine which isoform of CPT I is inhibited during endotoxaemia and whether the inhibition is due to increased tyrosine nitration. Cardiac mitochondria were isolated from endotoxaemic suckling rats. To determine whether M- or L-CPTI was inhibited, we carried out titrations with DNP-etomoxir-CoA. Slopes of the titration curves with DNPetomoxir-CoA were no different between control and endotoxaemia, suggesting that M-CPTI was specifically inhibited. Immunoprecipitation was carried out using an anti-nitrotyrosine antibody. Immunoprecipitated proteins were identified by Western blotting with L- and M-CPTI specific antibodies. L-CPTI was nitrated both in control and in 2- and 6-h endotoxaemia mitochondria but there was no significant difference in the level of nitration. M-CPTI was also nitrated in control mitochondria but nitration was significantly increased at both 2- and 6h endotoxaemia. Either 10 mM 3-nitrotyrosine plus 40 Ag nitrated-albumin or 0.5 M dithionite, during immunoprecipitation, greatly decreased immunopositivity for M- and L-CPTI on WB. M-CPTI appears to be a novel target for peroxynitrite during endotoxaemia, which would alter myocardial substrate selection. D 2004 Elsevier B.V. All rights reserved. Keywords: Sepsis; Fatty acid oxidation; Carnitine palmitoyl transferase I; Nitration; Peroxynitrite

1. Introduction The effects of endotoxaemia or sepsis on myocardial substrate utilisation are unclear: fat is often considered to be a preferential fuel during sepsis [42] but whole body fatty acid oxidation per se may still be inhibited relative to the circulating hypertriglyceridaemia [35]. Fatty acid oxidation can be decreased [9,10,25,34,37,40], unaltered [24,29,43] or increased [32,44,45] during sepsis, but the results obtained in these studies are dependent on the species studied, the severity of sepsis or endotoxaemia and the method used for measurement of fatty acid oxidation. Fat is an essential fuel in infancy, such that suckling rats have a respiratory quotient close to 0.7 [5,18]. During endotoxaemia in suckling rats, whole body fat oxidation is decreased [18] as is the activity of heart carnitine palmitoyl transferase I (CPT I), whereas kidney CPT I activity is unaltered [16]. This decreased myocardial CPT I activity

was not due to alterations in the amount of immunoreactive protein [16]. As heart contains both muscle and liver isoforms of CPT I, whereas kidney contains only the liver isoform [6], we hypothesised that specific inhibition of the muscle isoform of CPT I during endotoxaemia was responsible for the difference in inhibition observed in kidney and heart. Superoxide, nitric oxide and peroxynitrite were all able to inhibit CPT I activity in isolated heart mitochondria [16]. Peroxynitrite-mediated nitration of protein tyrosine residues has been suggested to be important in modification of cardiac function during inflammation, endotoxaemia and oxidative stress [1,13,21,22,27,28,36], so we also investigated the possibility that CPT I is nitrated during endotoxaemia in suckling rats.

2. Materials and methods 2.1. Model of neonatal endotoxaemia

* Corresponding author. Tel.: +44-20-7905-2158; fax: +44-20-74046181. E-mail address: [email protected] (S. Eaton). 1388-1981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2004.03.006

The study was approved under the United Kingdom Home Office regulations for Animals (Scientific Proce-

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dures) Act 1986. Endotoxaemia was modelled in peak suckling Wistar rats (11– 13 days, mixed gender) by intraperitoneal injection of 300 Ag/kg of 12.5 mg/l lipopolysaccharide (LPS) (E. coli 055:B5, Sigma, Poole, Dorset, U.K.). One litter of 10 rats was split into two groups, five control rats and five endotoxic. Controls were injected with isovolaemic normal saline. Rectal temperature was measured using a digital probe. 2.2. Mitochondrial isolation and CPT I assay Two or six hours after injection of saline or LPS, rats were killed by cervical dislocation, the hearts were removed and placed in ice-cold medium A (120 mM KCl, 20 mM HEPES, 5 mM MgCl2, 1 mM EGTA, 5 mg/ml fatty acidfree bovine serum albumin (BSA)) pH 7.4, washed in this medium to remove blood, and a single mitochondrial preparation made from each group of five animals after homogenisation and differential centrifugation [16]. CPT I activity was measured as previously described [16]. Dinitrophenyl-etomoxir-CoA was synthesised from dinitrophenyl-etomoxir by the mixed anhydride method [3].

completely selective so M-CPT I cannot be measured simply by assaying in the presence of an excess of DNP-etomoxir-CoA [48]. At low concentrations of DNPetomoxir-CoA, however, only L-CPT I should be inhibited [48]. We therefore carried out titrations of CPT I activity in control and endotoxic heart mitochondria with DNP-etomoxir-CoA, reasoning that if L-CPT I activity was inhibited by endotoxaemia, the slope of the inhibition curve would be steeper than control, whereas if M-CPT I were inhibited by endotoxaemia, the slopes of the DNPetomoxir-CoA inhibition curves would be similar. As shown in Fig. 1a, the curves were superimposable and the slopes not significantly different, suggesting that MCPT I is specifically inhibited during endotoxaemia. As expected, the titration curves for control and endotoxic kidney mitochondria were identical (results not shown), as kidney contains only the L-isoform and kidney CPT I activity was not inhibited by endotoxaemia.

2.3. Immunoprecipitation and Western blotting For the immunoprecipitation experiments, f 1-mg mitochondrial protein was resuspended in 1 ml 250 mmol/ l sucrose/2 mmol/l HEPES/0.1 mmol/l EGTA/0.5% v/v Triton X-100, pH 7.4 with protease inhibitors (Roche Biochemicals), incubated 30 min at 37 jC and 20-Al Protein l Protein A/G Agarose suspension (Oncogene) added. Samples were incubated 1 h on ice, centrifuged for 2 min and the supernatant removed. To the supernatant was added 4-Ag polyclonal anti-nitrotyrosine antibody (rabbit, Calbiochem), which was then incubated overnight at 4 jC with shaking. Thirty microliters of Protein A/G Agarose suspension was added and samples incubated 90 min on ice. After centrifugation, beads were washed 3  with 1-ml Western blot wash buffer, and subjected to Western blotting using anti-MCPT I and anti L-CPT I antibodies as previously described [16]. 2.4. Statistical analyses Prism 3 (Graphpad Software, San Diego, California, USA) was used for statistical analyses and calculation of IC50 values.

3. Results and discussion 3.1. Titration of CPT I activity with DNP-etomoxir-CoA and with malonyl-CoA DNP-etomoxir-CoA is much more inhibitory towards LCPT I than towards M-CPT I [48]; however, it is not

Fig. 1. (A) Titration of heart CPT I activity with DNP-etomoxir-CoA. Heart mitochondria were isolated from control and endotoxic rats and assayed for CPT I activity in the presence of different amounts of DNP-etomoxir-CoA. [o] control; [.] endotoxaemia. Mean F S.E., n = 15 per group. (B) Determination of IC50 for malonyl-CoA on CPT I activity in heart mitochondria from [o] control, [.] endotoxaemia. Mean F S.E., n = 13 per group.

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L-CPT I has a very much higher IC50 for malonyl-CoA than M-CPT I [30], so that if M-CPT I were inhibited by endotoxaemia, the overall IC50 of heart mitochondria for malonyl-CoA should increase. This proved to be the case, in that the apparent IC50 of heart mitochondria for malonylCoA was increased from 2.3 Amol/l in control to 9.2 Amol/ l in endotoxaemia (Fig. 1b). 3.2. Nitration of CPT I Peroxynitrite, which is spontaneously generated from the reaction of superoxide with nitric oxide, has been shown to be involved in myocardial dysfunction induced by cytokines [13,36] and endotoxaemia [21,22]. Peroxynitrite nitrates both free and protein-incorporated tyrosine to 3-nitrotyrosine, and tyrosine nitration of specific proteins, such as manganese superoxide dismutase and succinyl-CoA:3-oxoacid CoA transferase, has been demonstrated during inflammation and oxidative stress [1,27,28]. As we have previously demonstrated that the endotoxaemia-induced decrease in CPT I activity is not due to decreased total immunoreactive protein [16] and that CPT I activity could be inhibited by peroxynitrite [16], we hypothesised that nitration of M-CPTI during endotoxaemia could account for its decreased activity. In order to investigate this possibility, we developed an immunoprecipitation and Western blotting protocol based on a previously described immunoprecipitation protocol for nitrotyrosine-containing proteins [26] but Western blotting using specific antibodies against L- and MCPT I rather than anti-nitrotyrosine antibodies. The results of typical immunoprecipitation experiments using L- and M-CPT I antibodies are shown in Fig. 2. Immunoprecipitations with an anti-nitrotyrosine antibody of mitochondria from control (Fig. 2A, lanes 2 and 4) and endotoxaemic (Fig. 2A, lanes 3 and 5) rats, followed by Western blotting with an antibody against M-CPT I, indicated a band corresponding to non-immunoprecipitated mitochondria (Fig. 2A, lane 1) which was increased in amount by endotoxaemia (i.e. lane 2 vs. 3, lane 4 vs. 5). Western blotting with an antibody against L-CPT I indicated a band present in immunoprecipitated mitochondria from control (Fig. 2B, lanes 2 and 4) and endotoxaemic (Fig. 2B, lanes 3 and 5) rats that was also present in the non-immunoprecipitated mitochondrial sample which was not altered in amount by endotoxaemia. Pre-incubation of anti-nitrotyrosine antibody with 10 mM 3-nitrotyrosine plus 40-Ag nitrated bovine serum albumin (Alexis Chemical Corp.) before the overnight incubation with mitochondria, or pretreatment of mitochondria with 0.5 M dithionite, which converts 3-nitrotyrosine to 3-aminotyrosine [26], gave decreased, but not completely abolished, immunoreactive MCPT I upon Western blotting (Fig. 2C). In order to quantify the amount of nitrated M- and L-CPT I, we carried out densitometric analysis of the blots, normalising for the amount of citrate synthase activity of the starting mitochondrial suspension. Results indicated that nitration of M-CPT I

Fig. 2. Western blotting for M- and L-CPT I following immunoprecipitation of nitrotyrosine-containing proteins from cardiac mitochondria. (A) Western blotting for M-CPT I. Lane 1: mitochondria (no immunoprecipitation). Lanes 2 – 5: mitochondria from after immunoprecipitation. Lanes 2, 4: control mitochondria, lanes 3, 5: endotoxic mitochondria. (B) Western blotting for L-CPT I. Lanes 1 – 5 as in A above. (C) Blocking of nitrotyrosine immunoreactivity. Lanes 1 + 2, immunoprecipitation, no blocking. Lanes 3 + 4, blocking with 10mM 3-nitrotyrosine plus 40-Ag nitrated bovine serum albumin. Lanes 5 + 6, incubation with 0.5M dithionite. Lanes 1, 3, 5: control mitochondria; lanes 2, 4, 6: endotoxic mitochondria, Western blotting for M-CPT I.

significantly increased during endotoxaemia, but nitration of L-CPT I was unaltered (Fig. 3). Results were similar when normalized for the starting amount of mitochondrial protein instead of citrate synthase (results not shown). By comparison of the density of bands of non-immunoprecipitated Western blots with immunoprecipitations of the same mitochondrial preparation, we calculated that 6.0% of M-CPT I was nitrated under control conditions, and this increased to 10.0% during endotoxaemia, although these calculations are clearly approximate. The finding that some M- and L-CPT I was apparently nitrated even in mitochondria isolated from control animals was unexpected. In previous experiments that have demonstrated nitration of specific proteins, basal levels of nitration in control conditions were minimal [1,27,28]. This apparent nitration may be an artifact generated during processing of

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Fig. 3. Immunoprecipitation of nitrated M- and L-CPT I from heart mitochondria. Mitochondria from control or endotoxic mitochondria were immunoprecipitated and analysed by Western blotting as described in Materials and methods. Results were analysed by densitometric scanning, normalising for citrate synthase activity. (A) Western blotting for M-CPT I. n = 15 – 23, *P = 0.001, **P < 0.0001 vs. control. (B) Western blotting for L-CPT I. n = 10 – 14, P = not significant.

samples, although it could be almost completely blocked by nitrated-BSA plus 3-nitrotyrosine or by incubation with dithionite. Further experiments are necessary to determine whether M- and L-CPT I are nitrated under normal conditions; however, we believe that we have strong evidence for an increase in M-CPT I tyrosine nitration during endotoxaemia, which parallels decreases in M-CPT I activity. Peroxynitrite is produced from the reaction of superoxide and nitric oxide, and has been shown to react with several mitochondrial proteins in including aconitase, complexes I, II and IV, cytochrome c, adenine-nucleotide translocase, creatine kinase, manganese superoxide dismutase, and glutathione peroxidase [39]. M-CPT I could react with peroxynitrite produced in the cytosol, endothelium, or intramitochondrially from the reaction of nitric oxide from mitochondrial nitric oxide synthase [12] with superoxide derived from the respiratory chain. M-CPT I is a transmembrane protein of the outer mitochondrial membrane, so further speculation on the source of the peroxynitrite awaits identification of tyrosine residues modified during endotoxaemia and their location vis a vis the outer

membrane. Depending on the source of the peroxynitrite, some intramembrane tyrosine residues can be nitrated [50]. Using specific L- and M-CPT I antibodies, we have shown tat M-CPT I is a novel target for peroxynitrite during endotoxaemia, in addition to known targets [39]. A proteomic approach, such as that already undertaken in other models of inflammation [1], may be necessary to identify other targets of peroxynitrite-mediated nitration during endotoxaemia. Peroxynitrite production and/or nitrotyrosine formation has been shown to be increased in the endotoxaemic or septic heart [15,21 – 23] and has been suggested to contribute to contractile dysfunction [13 – 15]; however, the effects of increased peroxynitrite generation on substrate oxidation in the intact heart are unknown. The heart obtains 75% of its energy requirements from fatty acids under normal conditions [33] but uses other fuels such as glucose, lactate and ketone bodies [46]. Our results would lead one to predict that endotoxaemia or peroxynitrite would cause a decrease in myocardial fatty acid oxidation. However, succinylCoA:3-ketoacid-CoA transferase, a mitochondrial enzyme of ketone body utilisation [49], is known to be nitrated and inhibited by endotoxaemia [28] and it is likely that aconitase of the Krebs cycle is also inhibited by peroxynitrite during sepsis [7]. In addition, endotoxaemia and sepsis affect respiratory chain activity in the heart of adult rabbits and baboons [20,47] and suckling rats in the same model as used in the current study [17]. Therefore, it is difficult to predict the overall effects of systemic inflammation and sepsis on substrate utilisation. Perfused heart studies are required to determine the full effects of endotoxaemia in suckling rats on myocardial substrate utilisation. In addition, peroxynitrite is not the only route by which tyrosine residues can become nitrated; heme-containing peroxidases can also promote tyrosine nitration during inflammation in the presence of nitrite [19] so that the nitration of CPT I cannot be simply ascribed to peroxynitrite. These studies were undertaken in suckling rats of mixed gender, and therefore potentially missed important differences in the response to sepsis. The response to sepsis differs with gender [2], probably due to differences in the inflammatory cascade. There is little information in the literature available concerning the effect of gender on CPT I activity: hepatic CPT I activity is greater in male than female rats [41] and whereas there is no difference in muscle CPT I activity between human males and females [4], total CPT activity is higher in trained male than female humans [8]. In PPARa-deficient mice, males are much more likely than females to suffer from cardiac and hepatic lipid accumulation and sudden death, suggesting gender-related differences in the regulation of cardiac fatty acid metabolism [11]. Many advances in understanding the relationship of primary structure to catalysis of L- and M-CPT I have recently been made, and although the structure of CPT I has not been directly determined, a structural model of L-

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CPT I has been recently been generated based on the structure of carnitine acetyltransferase [31]. In this model, a tyrosine residue, Tyr589, which is conserved between species and in M-CPT I, forms a hydrogen bond with carnitine and is thus involved in catalysis and nitration could potentially impair enzymatic activity. However, this amino acid is also present in L-CPT I, for which we found no evidence of increased nitration in the current study. Further studies, such as identification of nitrated residues by MS/MS or MALDI-TOF [38], are necessary to clarify the molecular basis of these observations.

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4. Conclusions [17]

Heart M-CPT I is inhibited during neonatal endotoxaemia and this appears to be partially due to increased nitration of tyrosine residues. Such changes are predicted to alter myocardial substrate selection during sepsis although, as other mitochondrial functions are altered during sepsis, it is difficult to predict the precise in vivo effects.

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