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. 2012;7(9):e44383.
doi: 10.1371/journal.pone.0044383. Epub 2012 Sep 18.

Obese rats exhibit high levels of fat necrosis and isoprostanes in taurocholate-induced acute pancreatitis

Javier Pereda  1 , Salvador Pérez, Javier Escobar, Alessandro Arduini, Miguel Asensi, Gaetano Serviddio, Luis Sabater, Luis Aparisi, Juan Sastre
Affiliations

Obese rats exhibit high levels of fat necrosis and isoprostanes in taurocholate-induced acute pancreatitis

Javier Pereda et al. PLoS One. 2012.

Abstract

Background: Obesity is a prognostic factor for severity in acute pancreatitis in humans. Our aim was to assess the role of oxidative stress and abdominal fat in the increased severity of acute pancreatitis in obese rats.

Methodology: Taurocholate-induced acute pancreatitis was performed in lean and obese Zucker rats. Levels of reduced glutathione, oxidized glutathione, L-cysteine, cystine, and S-adenosylmethionine were measured in pancreas as well as the activities of serine/threonine protein phosphatases PP1 and PP2A and tyrosin phosphatases. Isoprostane, malondialdehyde, triglyceride, and free fatty acid levels and lipase activity were measured in plasma and ascites. Lipase activity was measured in white adipose tissue with and without necrosis and confirmed by western blotting.

Findings: Under basal conditions obese rats exhibited lower reduced glutathione levels in pancreas and higher triglyceride and free fatty acid levels in plasma than lean rats. S-adenosyl methionine levels were markedly increased in pancreas of obese rats. Acute pancreatitis in obese rats led to glutathione oxidation and lower reduced glutathione levels in pancreas together with decreased activities of redox-sensitive phosphatases PP1, and PP2A. S-adenosyl methionine levels decreased but cystine levels increased markedly in pancreas upon pancreatitis. Acute pancreatitis triggered an increase in isoprostane levels in plasma and ascites in obese rats. Free fatty acid levels were extremely high in pancreatitis-associated ascitic fluid from obese rats and lipase was bound with great affinity to white adipose tissue, especially to areas of necrosis.

Conclusions: Our results show that oxidative stress occurs locally and systemically in obese rats with pancreatitis favouring inactivation of protein phosphatases in pancreas, which would promote up-regulation of pro-inflammatory cytokines, and the increase of isoprostanes which might cause powerful pulmonary and renal vasoconstriction. Future studies are needed to confirm the translational relevance of the present findings obtained in a rat model of taurocholate-induced pancreatic damage and necrosis.

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Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflicts: Juan Sastre is a PLOS ONE Editorial Board member. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Pancreatic GSH and GSSG levels in lean and obese rats with acute pancreatitis.
Pancreatic levels of GSH (A) and GSSG (B) in lean and obese (Zucker) rats at 0, 1 and 6 hours after induction of acute pancreatitis. The number of rats per group was 8 for lean rats sacrificed at 0 or 6 h and for obese rats sacrificed at 0 h, 10 for obese rats sacrificed at 6 h, and 4 for lean and obese rats sacrificed at 1 h. The statistical difference is indicated as follows: * P<0.05 vs. time “0”. # P<0.05 in obese vs. lean in the same conditions.
Figure 2
Figure 2. Hepatic and pulmonary GSH levels in lean and obese rats with acute pancreatitis.
Levels of GSH in liver (A) and in lung (B) in lean and obese (Zucker) rats at 0 and 6 hours after induction of acute pancreatitis. The number of rats per group was 8. The statistical difference is indicated as follows: ** P<0.01 vs. time “0”.
Figure 3
Figure 3. Characterization of the transulfuration pathway in the pancreas in acute pancreatitis.
Levels of methionine (A), S-adenosyl methionine (B), cysteine (C) and cystine (D) in lean and obese (Zucker) rats at 0, 1 and 6 hours after induction of acute pancreatitis. The number of rats per group was 8 for lean rats sacrificed at 0 or 6 h and for obese rats sacrificed at 0 h, 10 for obese rats sacrificed at 6 h, and 4 for lean and obese rats sacrificed at 1 h. The statistical difference is indicated as follows: * P<0.05 and ** P<0.01 vs. time “0”. # P<0.05 and ## P<0.01 in obese vs. lean in the same conditions.
Figure 4
Figure 4. Phosphatase activity in lean and obese rats at 0 and 6 hours after induction of acute pancreatitis.
Serine/threonine protein phosphatases activity -PP1 (A) and PP2A (B)- and tyrosin phosphatases (PTPs) activity (C) are shown as release of PO4 −2 per min. Results are normalized by miligrams of protein. The number of rats per group was 6 for lean rats sacrificed at 0 or 6 h, and 7 for obese (Zucker) rats sacrificed at 0 h or 6 h. The statistical difference is indicated as follows: ** P<0.01 vs. time “0”.
Figure 5
Figure 5. Lipid peroxidation in plasma and ascitic fluid from lean and obese rats with acute pancreatitis.
Levels of MDA and 15-isoprostane F2t in plasma (A,C) and ascitic fluid (B,D) in lean and obese (Zucker) rats at 0 and 6 hours after induction of acute pancreatitis. The number of rats per group was 8 for determinations in plasma from lean and obese rats at 0 or 6 h and in ascitic fluid from obese rats at 6 h; 4 for determinations in ascitic fluid from lean rats at 6 h. The statistical difference is indicated as follows: * P<0.05 and ** P<0.01 vs. time “0”. # P<0.05 and ## P<0.01 obese vs. lean in the same conditions.
Figure 6
Figure 6. Lipid profile in plasma and ascitic fluid from lean and obese rats with acute pancreatitis.
Concentration of triacylgricerides (TAG) and free fatty acids (FFA) in plasma (A,C) and ascitic fluid (B,D) in lean and obese (Zucker) rats at 0, 1 and 6 hours after induction of acute pancreatitis. The number of rats per group was 8 for determinations in plasma from lean and obese rats at 0 or 6 h and in ascitic fluid from obese rats at 6 h; 4 for determinations in plasma from lean and obese rats at 1 h and in ascitic fluid from lean rats at 6 h. The statistical difference is indicated as follows: * P<0.05 and ** P<0.01 vs. time “0”. # P<0.05 and ## P<0.01 obese vs. lean in the same conditions. † P<0.05 and †† P<0.01 obese 6 h vs. obese 1 h.
Figure 7
Figure 7. Fat necrosis and pancreatic lipase in abdominal white adipose tissue from lean and obese rats with acute pancreatitis.
Macroscopic quantification of fat necrosis in lean and obese (Zucker) rats at 0 and 6 hours after induction of acute pancreatitis (A). Presence of pancratic lipase in adipose tissue in acute pancreatitis is illustrated by the increase of pancreatic lipase activity in white adipose tissue (WAT) and fat necrosis (FN) (B). A representative image of the presence of pancreatic lipase in WAT and FN is shown (C). Erk 1/2 was used as loading control. The number of rats per group was 8–10 for A and B, and 6–9 for C. The statistical difference is indicated as follows: ** P<0.01 vs. time “0”. ## P<0.01 obese vs. lean in the same conditions. † P<0.05 fat necrosis vs. WAT at 6 hours in obese rats.
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Grants and funding

This work was supported by grants (SAF2009-09500, SAF2006-06963, and CSD-2007-00020) from the Spanish Ministry of Science and Innovation together with FEDER funds to JS and grant UV-INV-AE11-42462 from the University of Valencia to JP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.