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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 1, 2002 as doi:10.1096/fj.01-0752fje. |
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Division of Gastroenterology and Liver Diseases, USC Liver Disease Research Center, Keck School of Medicine USC, Los Angeles, California, USA;
* USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, VA Greater Los Angeles Healthcare System, University of California, Los Angeles, California, USA;
Division of Hepatology and Gene Therapy, University of Navarra School of Medicine, Pamplona, Spain; and
Institute of Metabolic Disease, Baylor University Medical Center, Dallas, Texas, USA
2Correspondence: Division of GI and Liver Diseases, HMR Bldg., 415, Dept. of Medicine, Keck School of Medicine USC, 2011 Zonal Ave., Los Angeles, CA, 90033, USA. E-mail: shellylu{at}hsc.usc.edu
SPECIFIC AIMS
In mammals, two genes, MAT1A and MAT2A, encode for methionine adenosyltransferase (MAT), which catalyzes the formation of S-adenosylmethionine (SAM), the principal biological methyl donor and precursor for polyamines. The aims of the current study were to characterize MAT expression in normal pancreas and during pancreatic injury and to assess the role of SAM in two experimental models of pancreatitis.
PRINCIPAL FINDINGS
1. MAT1A is highly expressed in normal pancreas and pancreatic acinar cells
MAT1A and its encoded protein 
1, long thought to be expressed only in the liver, are highly expressed in normal rat and mouse pancreas (Fig. 1
). We found RNA expression of MAT1A and MAT2A in isolated rat pancreatic acini. Normal pancreas expresses predominantly MAT1A, as judged by Northern and Western blot analyses, and little MAT2A.
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2. Pancreatic MAT expression undergoes dramatic changes after feeding young female mice a choline-deficient and ethionine-supplemented (CDE) diet
Dramatic changes in pancreatic MAT expression are observed with the CDE diet and occurred by different mechanisms. 1 disappeared by 48 h on the CDE diet (Fig. 1)
despite nearly normal MAT1A mRNA levels. MAT2A mRNA and its protein levels were greatly induced (Fig. 1)
. In a more acute model of pancreatic injury (cerulein infusion in the rat), where pancreatic injury occurs within 6 h of treatment, there was no significant change in pancreatic MAT expression.
3. Pancreatic SAM levels fell after the CDE diet
Pancreatic SAM level (nmol/mg protein) in normal young (5-wk-old) female mice was 0.54 ± 0.01. This fell to 0.27 ± 0.02 (mean±SD from 2 pooled samples) and 0.30 ± 0.04 by 48 and 72 h of CDE diet, respectively (mean±SE from 4–5 samples for control and 72 h, P<0.05 vs. control). In old (14-month) female mice, pancreatic SAM level fell by 50% despite being resistant to pancreatitis.
Pancreatic SAM level (nmol/mg protein) in normal male rats treated with intravenous (i.v.) saline (control) was 0.41 ± 0.03 (mean±SE from 3 animals). It decreased to 0.32 ± 0.03 in rats treated with i.v. cerulein (mean±SE from 7 animals, P=0.07 vs. control).
Female MAT1A knockout mice had a much lower pancreatic SAM level than their wild-type (WT) littermates (WT=0.48±0.01, heterozygotes=0.21±0.005, homozygotes=0.08±0.01 nmol/mg, mean±SE from 3 animals in each group, P<0.05 vs. WT by ANOVA).
4. SAM supplementation prevented pancreatic injury in the CDE model and blunted pancreatic injury in the cerulein model
CDE diet in young female mice resulted in dramatic histological changes in the pancreas. The changes included a marked inflammatory response as well as necrosis and loss of parenchymal tissue (Fig. 2
). These histological changes were associated with an increase in the serum level of the pancreatic digestive enzyme amylase, which reached 6.4-fold at 72 h of CDE diet. Cerulein infusion resulted in an 11.2-fold increase in amylase levels. To see whether SAM supplementation prevents pancreatic injury, CDE- and normal diet-fed mice were treated with SAM at 15 mg/kg i.p. every 24 h. In the cerulein pancreatitis model, rats received SAM at 5 or 15 mg/kg i.v. 10 min before cerulein or saline (control) infusion. SAM supplementation in the CDE mice prevented pancreatic injury histologically (Fig. 2)
and biochemically. In the rat cerulein model, SAM supplementation at 5 mg/kg and 15 mg/kg resulted in attenuation of the cerulein-induced increase in serum amylase in rats (40% inhibition). Even stronger inhibition (55%) was observed for the cerulein-induced increase in serum lipase level. However, the protection was short-lived and no difference in amylase levels was found by 3 h after the termination of cerulein infusion.
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CONCLUSIONS AND SIGNIFICANCE
Evidence is accumulating that in the liver, a switch in MAT expression can affect SAM level and decreased SAM level can predispose to injury. The reason a switch from MAT1A to MAT2A expression can result in lowering of hepatic SAM level is due to the different kinetic profiles and regulatory properties of the MAT isozymes. The Km for methionine is lowest for MAT II (
4–10 µM), intermediate for MAT I (23 µM-1 mM), and highest for MAT III (215 µM-7 mM), with different studies reporting different absolute values. The activity of MAT is modulated by SAM, the product of the reaction it catalyzes. SAM strongly inhibits MAT II (IC50=60 µM, which is close to the normal intracellular SAM concentration), but minimally inhibits MAT I (IC50=400 µM) and stimulates MAT III (up to 8-fold at 500 µM SAM concentration). Because of these differences, the type of MAT expressed by a cell can influence the cell’s steady-state SAM level and methylation status. Thus, the SAM level in cells that express only the MAT II isoform should be relatively unaffected by fluctuations in methionine availability. In contrast, the rate of SAM synthesis and SAM level increased with increasing methionine availability in cells that express liver-specific MAT. Using a cell line model that differs only in the type of MAT expressed, we showed that cells expressing MAT1A had the highest intracellular SAM level and DNA methylation whereas cells that expressed MAT2A had the opposite.
In the liver, absence of MAT1A and chronic SAM depletion led to spontaneous oxidative stress, development of steatohepatitis, and hepatocellular carcinoma, whereas SAM supplementation protects against various forms of liver injury. Our finding that MAT1A is highly expressed in the normal pancreas suggests that observations we made in the liver may apply to the pancreas. Whether a decrease in SAM level would predispose the organ to injury is intriguing. However, our data show that results on the role of MAT1A and SAM in the liver cannot be extrapolated to the pancreas. A CDE model has been used extensively to study the pathogenesis of pancreatitis but the mechanism of pancreatitis in this model remains unclear. Only young female mice are susceptible to pancreatitis with this diet. A choline-deficient diet is known to result in depletion of hepatic SAM levels. Since pancreatic MAT expression mirrors that in the liver, we expected pancreatic SAM levels to fall in the CDE model. Indeed, we found that pancreatic SAM level fell 50% after 48 h on the CDE diet. The fall in SAM level can be attributed partly to a dramatic switch in MAT expression. MAT1A-encoded protein disappeared after 48 h on the CDE diet whereas MAT2A-encoded protein was greatly induced. Whether MAT activity was inhibited is unclear and requires further study. Our findings indicate that changes in pancreatic MAT isozymes are mediated by different mechanisms. In the case of MAT1A, it appeared to be a post-translational effect since there was no significant decrease in its mRNA level. We had previously observed a similar dissociation between hepatic MAT1A mRNA and 1 protein levels in two other models after treatment with thioacetamide and intragastric ethanol. Thus, it is possible that the stability of MATI/III becomes impaired after certain experimental treatments.
In the young female mice fed the CDE diet, the fall in pancreatic SAM level may play a role in the development of pancreatitis. This is supported by the fact that SAM supplementation almost completely prevented the pancreatic injury. Previous studies have shown that methionine supplementation protected young female mice against CDE-induced pancreatitis. Methionine is catabolized to SAM in all cells, so that the protective effect may have been mediated via SAM. However, a fall in SAM level alone cannot explain development of pancreatitis after the CDE diet since old female mice resistant to this form of injury showed a similar fall in pancreatic SAM level on this diet. MAT1A knockout mice have a profound reduction in pancreatic SAM level (83% lower) compared to WT controls but exhibit no pancreatic injury. Thus, it is unclear how SAM supplementation protects against CDE-induced pancreatitis in young female mice. Factors influenced by age and gender are clearly important in determining the susceptibility of the animal to CDE-induced pancreatitis. The interaction of SAM and these factors remains to be determined. In the liver, decreased SAM level can potentiate the TNF- response. This is supported by studies showing that rats with decreased hepatic SAM levels were predisposed to liver injury caused by lipopolysaccharide, which was prevented with exogenous SAM treatment. Since NF-
B activation and TNF- up-regulation play important roles in the pathogenesis of pancreatitis, whether SAM modulates these pathways in acinar cells and whether they are age and/or gender dependent will be important to examine.
We next studied the rat cerulein pancreatitis, where few changes in SAM or MAT protein levels were observed. This may reflect the fact that the animals were treated with the agent i.v. for 6 h before being killed. Even so, SAM supplementation blunted the injurious effect of cerulein. SAM’s effect was not due to alteration of the CCK receptor signaling since CCK-induced amylase secretion was unaffected. Although SAM was protective immediately after cerulein infusion, its protective effect disappeared 3 h after the end of cerulein infusion. Similar to the CDE-induced pancreatitis model, the mechanism of SAM’s protection is unclear.
In summary, the current work presents several novel findings. 1) MAT1A, thought to be a liver-specific gene, actually is highly expressed in normal rat and mouse pancreas, particularly in the pancreatic acinar cell. 2) Dramatic changes in MAT expression occur in the CDE pancreatitis model and by different mechanisms. 3) SAM supplementation prevented pancreatic injury in the CDE model and blunted pancreatic injury in the cerulein model. 4) Despite the protective effect of SAM, SAM depletion alone does not result in pancreatic injury. Collectively, these data support the notion that SAM may regulate the inflammatory and cell death responses in the pancreas in some models of injury, but its effects may be quite different from SAM’s generalized protective action in the liver. Fig. 3
summarizes the main findings of this work.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0752fje; to cite this article, use FASEB J. (November 1, 2002) 10.1096/fj.01-0752fje 
VA Greater Los Angeles Healthcare System, 11301 Wilshire Blvd., Bldg. 258 Room 340, Los Angeles, CA 90073. E-mail: Stephen.Pandol@med.va.gov
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