|Published (Last):||20 May 2005|
|PDF File Size:||12.41 Mb|
|ePub File Size:||5.50 Mb|
|Price:||Free* [*Free Regsitration Required]|
Acute pancreatitis was induced by cerulein in mice, and a group of animals received S-adenosylmethionine treatment. Protein steady-state levels of S-adenosylhomocysteine-hydrolase and cystathionine gamma-lyase diminished but methylthioadenosine phosphorylase levels increased in pancreas with acute pancreatitis.
Accordingly, S-adenosylmethionine administration increased inflammatory infiltrate and edema in pancreas with acute pancreatitis. Methionine is an essential sulfur amino acid that is not only involved in protein biosynthesis, but it also acts as a metabolic precursor for critical metabolic pathways [ 1 , 2 ]. It is the first precursor for synthesis of S-adenosylmethionine SAM , the principal biological methyl donor in cells.
Through this metabolic pathway, SAM donates its methyl group to a large variety of acceptors molecules including nucleic acids, proteins, and lipids. S-adenosylhomocysteine SAH , the product of all trans-methylation reactions, is converted into homocysteine and next, homocysteine is remethylated into methionine to allow methionine recycling in cells [ 3 , 4 ].
On the other hand, using homocysteine as intermediary and through the trans-sulfuration pathway, methionine can also act as the primary source of cysteine, a limiting factor for the synthesis of reduced glutathione GSH [ 1 ]. Hence, methionine metabolism through the trans-sulfuration pathway and the trans-methylation system decisively contributes to the maintenance of redox homeostasis in cells [ 6 ].
Increased levels of homocysteine, a sign of dysregulation of the trans-sulfuration pathway, have been detected in plasma of patients with a variety of gastrointestinal disorders including inflammatory bowel disease [ 7 , 8 ], colorectal cancer [ 9 ] as well as acute pancreatitis [ 10 ].
Acute pancreatitis AP is an inflammatory process of the pancreatic gland that may eventually lead to systemic complications [ 11 ]. Pancreatic GSH depletion is an early feature in the development of acute pancreatitis, and this depletion when maintained in a long term contributes to the severity of the disease [  ,  ,  ]. Furthermore, SAM levels decreased in pancreas during acute pancreatitis in rats [ 16 ].
Nevertheless, the precise mechanism involved in the dysregulation of the trans-sulfuration pathway in acute pancreatitis remains to be explored. In this work, firstly we have studied the methionine cycle as well as the trans-sulfuration pathway using metabolomic and proteomic approaches in an experimental model of acute pancreatitis identifying the causes of the dysregulation; secondly, we have detected the effects of SAM administration on these pathways.
Acute pancreatitis was induced in week-old mice. Mice received intraperitoneal injections of cerulein Sigma-Aldrich, St. The sacrifice was confirmed by cervical dislocation. The conditions employed were: positive electrospray ionization ESI , capillary voltage 3. A binary gradient was used in which mobile phase A was 0. These conditions were maintained for 1. Mass spectrometric detection was carried out by multiple reaction monitoring MRM employing the acquisition parameters summarized in Table 1.
Two MRM transitions per analyte were acquired for quantification and confirmation [ 18 ]. Total protein was determined using a colorimetric method. After digestion, samples were desalted using ZipTip Merck according to manufacturer instructions. When electrophoresis was performed under non-reducing conditions, the sample buffer had the same composition but without DTT.
Tissue extracts were prepared and subjected to immunoprecipitation with specific antibody against CBS as previously described [ 21 ]. The immunoprecipitates were assessed for the presence of nitro-tyrosine by western blotting with anti-nitro-tyrosine antibody.
The specific primers used are shown in Table 2. Tbp was used as housekeeping gene to normalize the transcription analysis. Chromatin from frozen pancreatic tissues was isolated as described in [ 22 ]. Statistical analysis was performed in two steps. When the overall comparison of groups was significant, differences between individual groups were investigated using the Bonferroni test.
In contrast, both pancreatic SAH and homocysteine levels remained unchanged during cerulein-induced acute pancreatitis Fig. The number of mice per group was 4—6. To assess if homocysteine metabolism was impaired through the trans-sulfuration pathway in acute pancreatitis, we measured cystathionine, cysteine, and glutathione levels in pancreas during cerulein-induced acute pancreatitis by mass spectrometry.
In pancreatic tissue, we analyzed by proteomics the steady-state protein levels of 10 enzymes involved in methionine cycle and trans-sulfuration pathway Fig. Pancreatic levels of S-adenosylhomocysteine-hydrolase SAHH and cystathionine gamma-lyase CGL , the enzymes that catalyze the conversion of SAH into homocysteine and cystathionine into cysteine, respectively, were reduced upon cerulein-induced acute pancreatitis Fig.
In contrast, the pancreatic expression of methylthioadenosine phosphorylase MTAP , a critical enzyme for the methionine salvage pathway, was increased in mice with cerulein-induced acute pancreatitis Fig. The number of mice per group was 6—8.
It has been reported that thiol oxidation as well as tyrosine nitration of CBS may compromise its enzymatic activity [ 24 , 25 ]. Thiol oxidation of CBS was not found in pancreas with acute pancreatitis Fig.
S1A , whereas CBS nitration was remarkably increased and accordingly Nos2 gene expression and its protein levels were markedly up-regulated in pancreas upon cerulein-induced acute pancreatitis Fig.
Supplementary Fig. S2 shows the general level of protein nitration in pancreatic tissue in mice with cerulein-induced acute pancreatitis.
Additionally, serine deficiency may also act as a limiting factor for CBS enzyme activity. However, pancreatic serine levels remained unchanged upon cerulein-induced acute pancreatitis Fig. In order to confirm the blockade of homocysteine metabolism during cerulein-induced acute pancreatitis, SAM was administered. The impairment of homocysteine re-methylation to methionine during acute pancreatitis was corroborated by measuring methionine levels in pancreatic tissue. In addition, treatment with SAM did not modify the pancreatic levels of cystathionine, cysteine, and GSH in SAM-treated mice with pancreatitis compared with those mice without treatment.
Thus, the levels of these metabolites remained low in these two experimental groups with pancreatitis compared with control mice Fig. We found that the promoter regions of these two genes were enriched in H3K4me3 in SAM-treated mice with pancreatitis in comparison with SAM-untreated mice with pancreatitis Fig. Histological analysis of the pancreatic tissue revealed that both inflammatory infiltrate and edema were more intense in SAM-treated mice with pancreatitis in comparison with SAM-untreated mice with pancreatitis Fig.
Nos2 gene expression was higher in SAM-treated mice with pancreatitis in comparison with untreated mice with pancreatitis Fig. In the present work, we show that nitrosative stress dysregulates methionine metabolism in pancreas in acute pancreatitis directly affecting the trans-sulfuration pathway.
Our results show a progressive depletion in pancreatic levels of methionine with parallel decrease in cystathionine and cysteine. SAM and glutathione levels were also rapidly depleted in pancreas during experimental acute pancreatitis in accordance with previous reports [ 16 , 17 ]. In this work, we have also found that pancreatic SAH and homocysteine levels remain unchanged upon cerulein-induced acute pancreatitis.
We have identified the cause of this dysregulation showing that tyrosine-nitration of CBS seems to be responsible for blockade of the trans-sulfuration in experimental acute pancreatitis. Therefore, in acute pancreatitis CBS nitration impairs homocysteine metabolism and limits metabolic flux through this pathway. CBS is a tetrameric redox-sensitive and rate-limiting enzyme responsible for homocysteine conversion to cystathionine.
Thus, the redox status of the heme cofactor has been reported as a central redox modulator of CBS activity.
Recently, it has been reported that a redox-active disulfide bond modulates CBS activity [ 25 ]. However, we have not found thiol oxidation of CBS in acute pancreatitis. It is noteworthy that the levels of tyrosine-nitrated CBS were markedly increased in this model of acute inflammation.
Nitration-mediated loss of CBS activity associated with elevated levels of homocysteine has been previously reported in aging rats [ 24 ].
In our model, CBS nitration impairs homocysteine metabolism through the trans-sulfuration pathway restraining GSH biosynthesis.
It is known that up-regulation of Nos2 expression triggers nitrosative stress in pancreas with acute pancreatitis [ 35 , 36 ] as it occurs generally in acute inflammation. Hence, pancreatic levels of nitrotyrosine, a marker of nitrosative stress, increased in mice with cerulein-induced acute pancreatitis [  ,  ,  ].
Our results confirm the increased Nos2 expression and show the detrimental effect of nitrosative stress on the trans-sulfuration pathway in acute pancreatitis, and specifically on CBS regulation.
Furthermore, in accordance with other studies our results suggest that Nos2 , but not Nos1 or Nos3 , is the main source of NO and the major contributor to nitrosative stress during acute pancreatitis [ 36 , 37 ]. In fact, Nos2 -deficient mice exhibited a reduced degree of pancreatic inflammation and tissue injury in pancreas with acute pancreatitis [ 37 ]. In contrast and strikingly, genetic deletion of Nos3 aggravated the severity of acute pancreatitis in mice [ 40 ]. Hence, here we are proposing that Nos2 up-regulation triggers CBS nitration and blockade in the metabolism of homocysteine in pancreas during acute pancreatitis.
CBS nitration might be widely associated with inflammation as this would explain the increased levels of homocysteine found in a variety of inflammatory disorders, including inflammatory bowel disease [ 7 , 8 ], and vascular inflammation [ 41 ]. Furthermore, the elevated levels of homocysteine would contribute to severity of inflammatory diseases promoting oxidative stress, endothelial dysfunction, and cardiovascular disease [  ,  ,  ]. CBS is also involved in the synthesis of hydrogen sulfide H 2 S together with cystathionine gamma-lyase CGL and mercaptopyruvate sulfurtransferase [ 44 ].
CGL is the major enzyme responsible for H 2 S synthesis in pancreas during pancreatitis [ 44 ] so the contribution of CBS in this regard is likely to be minor. The role of H 2 S in inflammation is still controversial as it may exert opposite effects on the inflammatory response depending on the tissue, its concentration and its source [  ,  ,  ,  ].
Endogenous H 2 S seems to exert a pro-inflammatory role in pancreatitis as GCL knock-out mice exhibited less pancreatic edema, inflammatory infiltrate, and acinar necrosis in acute pancreatitis [ 44 ].
CBS nitration may directly limit the ability of exogenous SAM to modulate the trans-sulfuration pathway and GSH levels in experimental acute pancreatitis. Consequently, homocysteine is accumulated in pancreas during acute pancreatitis upon SAM treatment. Although it is known that SAM supplementation exhibits hepatoprotective effects against liver injury and may provide beneficial effects ameliorating inflammation-induced colon cancer in mice [ 4 , 49 ], we show here that SAM treatment clearly exhibits a pro-inflammatory effect in acute pancreatitis in mice suggesting that SAM administration in acute inflammatory disorders may not be beneficial.
These findings suggest that SAM supplementation may increase histone methylation in the promoters of pro-inflammatory genes inducing their expression. Consequently, acute pancreatitis was aggravated upon SAM administration and indeed, we found more intense edema and inflammatory infiltrate in the pancreas of SAM-treated mice with pancreatitis. Our results highlight for the first time the adverse effects of nitrosative stress in acute inflammation through CBS nitration and impairment of homocysteine metabolism.
Hence, in this work we provide new insights to understand the mechanisms, not yet fully elucidated, that lead to increased levels of homocysteine in inflammatory disorders. Read article at publisher's site DOI : This data has been text mined from the article, or deposited into data resources. To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation. J Nutr , 8 , 01 Aug Cited by 44 articles PMID: Redox Rep , 8 1 , 01 Jan Cited by 54 articles PMID: Biochem Pharmacol , 65 9 , 01 May
Anexo:Fallecidos en abril de 2017
In , a total of papers were published in the journal, with a median time to first decision of 41 days and a median time from submission to publication of 22 days. The editors would like to express their sincere gratitude to the following reviewers for their generous contribution in National Center for Biotechnology Information , U. Journal List Animals Basel v. Animals Basel.
Mina y gigante
El árbol de las hojas pardas