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Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
2To whom correspondence should be addressed. E-mail: hara{at}chem.agr.hokudai.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • dietary amino acids • chymotrypsin • translation initiation • eukaryote initiation factor • rats
Amino acids (AA)2 are not only components, but also regulators of protein metabolism. That is, AA, as well as insulin, stimulate protein synthesis and suppress protein degradation in muscle (1–3). Recently, the regulatory effects of AA on translation initiation have been reported (4–10). Some AA stimulate translation initiation via the mammalian target of rapamycin (mTOR) but not insulin receptors (7), whereas insulin stimulates mTOR via phosphorylation of the tyrosine residues of its receptor (8). Furthermore, mTOR regulates eukaryote initiation factors (eIF) at translation initiation and protein synthesis in muscle (4), liver (5,9) and pancreatic beta cells (10). The formation of an eIF4F complex, consisting of eIF4A, eIF4E and eIF4G, plays a key role in translation initiation, and the availability of eIF4E is rate-limiting for the complex formation. Phosphorylation levels of eIF4E-binding protein (4E-BP) indicate the formation of eIF4F because low phosphorylated 4E-BP1 (called 
- or ß-form) inactivates eIF4E by binding to this factor and high phosphorylated 4E-BP1 (
-form) liberates eIF4E (4). Thus, the status of 4E-BP1 phosphorylation and the association of eIF4E with 4E-BP1 or eIF4F are thought to be indicators for the mTOR-pathway and translation initiation activity.
We have previously shown that dietary AA induce pancreatic proteases independently of cholecystokinin (CCK) in rats (11–13). Dietary protein induces pancreatic proteases via secretion of CCK, which is a potent pancreatic protease inducer (14–16), whereas AA do not stimulate CCK secretion in rats (17). These results suggest that there is an unknown AA-regulating mechanism for pancreatic protease induction. However, the regulatory effect of AA and/or insulin on translation initiation in the exocrine pancreas has not been reported.
The aims of the present study were to evaluate changes in the synthesis of pancreatic enzymes and mRNA levels induced by dietary AA, and to clarify the mechanism of the enhancement of pancreatic enzyme synthesis, especially that of eIF4-associating proteins at the translation initiation stage.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Wistar/ST rats (5-wk-old; Japan SLC, Hamamatsu, Japan) weighing 
100 g were used in this study. Rats were fed a sucrose-based AIN-76 semipurified basal diet during acclimation as reported previously (11). Four test diets were prepared with adjustment of the sucrose content in the basal diet to contain 20 and 60 g of casein per 100 g diet (20 and 60% diet, respectively). An AA-simulated casein, in which the nitrogen content was equal to that in casein, was also used (AA diet) as reported previously (11). All AA were obtained from Ajinomoto (Tokyo, Japan).
This study was approved by the Hokkaido University Animal Committee, and the rats were maintained in accordance with the guidelines of Hokkaido University for the care and use of laboratory animals.
Experimental procedures
In the first of two experiments (expt. 1), rats were maintained in a 12-h light/dark cycle (light period 800–2000h), ate the respective diets ad libitum and had free access to water. After acclimation with the basal diet for 3 d, rats were divided into 4 groups (n = 6 or 7) and given a 20% casein, 60% casein, 20% AA or 60% AA diet for 7 d. On d 7, 1.85 MBq [35S] methionine (Met) (Institute of Isotopes, Budapest, Hungary) was intravenously injected into all rats from 900 to 1130 h. Exactly 30 min later, two pieces of pancreas were excised under pentobarbital anesthesia to measure mRNA, [35S] Met incorporation into the pancreatic enzymes and a translation initiation factor. The rats were then killed by ensanguination from the abdominal aorta. The remainder of the pancreas was then removed, frozen quickly in liquid nitrogen and freeze-dried for subsequent evaluation of enzymatic activities.
In expt. 2, rats were maintained in a 0500–1700-h light cycle and had free access to water, but were only given the diets during the dark period. After acclimation with a basal diet for 3 d, rats were divided into two groups and given a 20 or 60% AA diet (same as in expt. 1) for 7 d. On d 7, rats were fed their respective diets at 1700 h (after food deprivation) and were then killed at 0, 1 or 3 h after feeding. Sample collection was the same as in expt. 1.
Analyses.
Immediately after removal, one segment of the pancreas (80 mg) was homogenized in 1 mL of ISOGEN (Nippon Gene, Tokyo, Japan) with a Polytron homogenizer (Kinematica, Amlehnhalde, Switzerland) for extraction of the total RNA and subsequent measurement of mRNA. Amylase, chymotrypsin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were quantified by Northern blot analysis using digoxigenin-labeled cDNA probes as previously described (12,18). The sets of sense and antisense primers for the synthesis of cDNA from the mRNA of amylase and GAPDH were previously reported (18) and those for chymotrypsin mRNA were as follows: 5'-TCAACGGAGAGGATGCTATT-3' and 5'-AGGAGTGGAAGTGGAACAGA-3'.
The other segment of the pancreas (50 mg) was homogenized in 1 mL of lysis buffer (pH 7.4; 10 mmol/L of HEPES, 5 mmol/L of EGTA, 1 mmol/L of EDTA, 10 mmol/L of MgCl2, 10 mmol/L of K2HPO4, 150 mmol/L of NaCl, 50 mmol/L of beta-phosphoglycerol, 2 mmol/L of phenylmethylsulfonyl fluoride, 1 mmol/L of benzamidine, 2 mmol/L of sodium orthovanadate, 100 mmol/L of NaF, 10 g/L of Triton X-100, 1 mg/L of leupeptin and 1 mg/L of aprotinin) with a Polytron homogenizer (Kinematica) for the measurement of protein synthesis and translation initiation factors.
Pancreatic enzyme synthesis activity was estimated by the incorporation of [35S] Met into the individual proteins separated by a 10–15% gradient of SDS-PAGE. Radioactivities in the individual bands were visualized and analyzed using a FUJIX Bas 1000 system (Fuji Photo Film, Tokyo, Japan).
The homogenate was immunoprecipitated with anti-eIF4E antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for analysis of the association of eIF4E with 4E-BP, eIF4G, and with anti-insulin receptor ß (IRß) antibody (Upstate Biology, Waltham, MA) for the IRß subunit. The immunoprecipitant was collected with Protein G-Plus agarose (Santa Cruz Biotechnology). The immunoprecipitant or homogenate for the estimation of 4E-BP1 status were resolved with a sample buffer (final concentration: 50 mmol/L of Tris-HCl, 200 g/L of SDS, 60 mL/L of 2-mercaptoethanol, 100 mL/L of glycerol) and subjected to immunoblot analysis and visualized by an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, Little Chalfont, England).
The dried pancreas was homogenized in a buffer solution (1.0 g/L of Triton X-100, 9.0 g/L of NaCl) and analyzed for protein content, and amylase and chymotrypsin activity as previously described (11).
Calculations.
Enzymatic activities were expressed in units per mg protein. The levels of mRNA were standardized for GAPDH mRNA, and expressed as a ratio to the 20% casein diet group (expt. 1) or the 20% AA diet group at 0 h (expt. 2). Enzyme synthesis activity was expressed as a percent of the intensity of each band compared with the total intensity of all bands. The phosphorylation status of 4E-BP1 was expressed as a ratio of the -form to the total intensity of the three bands (, ß and -form). Intensities of eIF4G and 4E-BP1 for association with eIF4E were normalized with the intensity of the eIF4E band. Phosphotyrosine (pY) level in IRß was estimated by normalization with the IRß band. All data are presented as means ± SEM.
Statistic analysis.
Data were analyzed by two-way ANOVA (StatView version 5.0 Software; SAS Institute, Cary, NC) and subsequent Duncan’s multiple range test. An unpaired Student’s t test was used to evaluate the differences in chymotrypsin mRNA levels between the two groups fed casein in expt. 1. We considered differences with P < 0.05 to be significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Diet chyme was found in the stomachs of all rats at death. Body weight gain and food intake in the AA diet groups were lower than in the casein diet groups, and pancreatic dry weight and protein content were higher in the 60% diet groups compared with the 20% diet groups (Table 1).
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-form (data not shown). The phosphorylation level of IRß in the 20% casein and AA diet groups were higher than in the 60% diet groups (Fig. 2).
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Body weight gain and food intake in the 60% AA diet group were lower than in the 20% AA diet group except at the 0 h point (Table 1). Pancreatic dry weight and protein content were higher in the 60% AA diet group compared with the 20% AA diet group.
Chymotrypsin activity was higher in the 60% AA diet group than in the 20% AA diet group at 0, 1 and 3 h after refeeding. In the 60% AA diet group, chymotrypsin activity increased with time, although there was no difference in the mRNA level between the 20 and 60% AA diet groups (Fig. 3A, B). In contrast, except for mRNA level at 0 h, pancreatic amylase activity and mRNA level were higher in the 20% AA diet group than in the 60% AA diet group throughout the experiment (Fig. 3C, D).
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-form of 4E-BP1 was observed in the either AA diet group at 0 h (Fig. 4A). In the 60% AA diet group, the percent of 4E-BP1 -form increased in a time-dependent manner after feeding compared with that at 0 h and was higher than in the 20% AA diet group at 3 h (Fig. 4A). In the 20% AA diet group, -form levels increased to a peak at 1 h and fell again at 3 h. Changes in eIF4G associated with eIF4E were similar to the 4E-BP1 status. The level at 3 h in the 60% AA diet group was double that at 0 h (Fig. 4B). On the other hand, the association of 4E-BP1 with eIF4E decreased after feeding either diet (P = 0.022, two-way ANOVA). In the 60% AA diet group, the association of 4E-BP1 with eIF4E at 3 h was lower than that at the 0 h point (P < 0.05; Fig. 4C).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In rats, dietary protein, but not AA, stimulates the secretion of CCK (17,19). CCK is a major factor in pancreatic protease induction associated with increases in pancreatic protease mRNA in rats (15). We have previously shown by using a potent CCKA receptor antagonist, devazepide, that CCK is not involved in protease induction after feeding a high AA diet (11). Another study using CCK-knockout mice also showed the CCK-independent induction of the pancreatic protease (20). These results indicate that AA play a regulatory role in pancreatic protease synthesis through a mechanism different than that of CCK. Dietary protein may influence protease induction both in the intestine, as protein or peptides, and after absorption, as AA. Chymotrypsin mRNA levels were higher in the 60% casein diet group than those in the 20% casein diet group (P = 0.088, Student’s t test; Fig. 1E). The protease induction caused by a high protein diet may be partly associated with CCK-independent increases in translational activities due to AA action in addition to CCK-dependent stimulations of transcription (15) and translation (21,22) in protein synthesis.
We determined the status of 4E-BP1 and the association of eIF4E with 4E-BP1 or eIF4G. Some AA regulate the translation initiation stage including eIF4 in muscle (4), liver (5,9) and pancreatic beta cells (10). The phosphorylation of 4E-BP1 to the -form promotes the formation of eIF4F and subsequent protein synthesis by releasing eIF4E (8). Activation of translation initiation is shown by the increase in the -form of 4E-BP1 and by the increase in the association of eIF4E with eIF4G or by the decrease in the association of eIF4E with 4E-BP1. In the present study, the -form of pancreatic 4E-BP1 increased in a time-dependent manner in rats fed the 60% AA diet (Fig. 4A). The association of 4E-BP1 with eIF4E decreased 3 h after feeding the 60% AA diet, as did 4E-BP1 phosphorylation status. However, ratios of eIF4E bound to eIF4G did not significantly increase in rats fed the 60% AA diet (P > 0.10, two-way ANOVA). The reason for this is unclear, but we speculate that eIF4E protein possibly increased when feeding the 60% AA diet on d 7 obscures differences among the ratio of each time-point, or eIF4G was degraded during the analytical process because of its sensitivity to breakdown. These changes, which coincided with those in chymotrypsin activity in rats fed the 60% AA diet, suggest that changes in the translation initiation factor are at least partly associated with the induction of chymotrypsin by a high AA diet. However, this was not observed in the rats fed the 20% AA diet (Figs. 3A,4A–C). There were no differences in 4E-BP1 status between groups at the early stage of the light period in expt. 1 (data not shown). In contrast, chymotrypsin synthesis was still higher in the 60% AA diet group than in the 20% AA diet group, which suggests that stimulation by the AA diet continued overnight (Fig. 1A, C). The discrepancy between the initiation activity and chymotrypsin synthesis in the later stage of feeding suggests that the activation of translation initiation factors, which are commonly thought to regulate the global synthesis of proteins (6), is not always associated with protease synthesis (5). The 60% AA diet stimulated synthesis of only the serine proteases, proelastase, chymotrypsinogen and trypsinogen (Fig. 2C, Table 2). The mechanism of specificity for serine proteases in induction with feeding of a high AA diet is not known. Possibly, mRNA encoding of these proteases has specific sequences influencing translation efficiency. However, this hypothesis does not explain the unchanged level of chymotrypsin activity at 1 h after feeding in the 20% AA diet group in expt. 2 (Figs. 3B, 4). Anther possible explanation is that the expression of mRNA species encoding chymotrypsinogen was different for the rats fed the 20 and 60% AA diet, or a structure of chymotrypsin mRNA was modified by d 7 feeding of the 60% AA diet. These differences or modifications of mRNA structure may alter its affinity to the eIF4F complex. Eukaryotic mRNA 5'-cap structure (caps 0, 1 or 2) are enzymatically modified (23), which relates to the affinity of mRNAs with initiation factors. Further studies should be done on the relationship between translation and modification of mRNA including methylation by mRNA (N6-adenosine)-methyltransferase (24,25).
Increased phosphorylation of 4E-BP1 in the early stages after feeding a high AA diet is transient as described above. Possibly, the changes in 4E-BP1 status are only necessary for the initial stages in the induction of protein synthesis.
We showed that pY in IRß was lower in rats fed both high casein and high AA diets (Fig. 2). The level of pY in IRß represents the intensity of insulin signals better than does plasma insulin level because insulin acts on acinar cells in a paracrine fashion (26,27). Insulin stimulates phosphorylation of tyrosine residues in IRß and leads to activation of downstream substances, mTOR, 4E-BP1 and eIF4F complex (8). The feeding of 20% casein and 20% AA diets, which are high carbohydrate diets, may stimulate 4E-BP1 phosphorylation via the insulin signaling pathway. We observed increased 4E-BP1 phosphorylation at 1 h in rats fed a 20% AA diet. On the other hand, AA stimulate mTOR, independently of phosphorylation of IRß, as described above. High levels of dietary AA may stimulate translation initiation independent of upper insulin signaling in the pancreas.
We also showed that amylase synthesis and enzymatic activity were lower in the high nitrogen diet groups (Figs. 1B, D, F, 3C, D). In contrast to chymotrypsin, these differences were associated with a large decrease in amylase mRNA levels. These results show that amylase synthesis is mainly regulated by diet at the mRNA level. Insulin is thought to regulate the mRNA level of pancreatic amylase (28,29). In both the 60% casein and 60% AA diet groups, a relative reduction in carbohydrates in the diet may have lowered the secretion of insulin (Fig. 2). Another possible explanation is that inhibition of insulin signaling by AA (30) reduces amylase mRNA levels.
In conclusion, dietary AA, as well as dietary protein, stimulate pancreatic protease synthesis. The protease induction, at least of chymotrypsin, to the AA level in diet is regulated in the translational stage. Furthermore, the eIF4 series at translation initiation may be partly involved in this regulation in the early stage of feeding of a high AA diet.
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FOOTNOTES |
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3 Abbreviations used: AA, amino acids; CCK, cholecystokinin; eIF, eukaryote initiation factor; 4E-BP1, eIF4E-binding protein 1; GAPDH, glycelaldehyde-3-phosphate dehydrogenase; IRß, insulin receptor ß; Met, methionine; mTOR, mammalian target of rapamycin; pY, phosphotyrosine.
Manuscript received 22 May 2003. Initial review completed 3 June 2003. Revision accepted 5 July 2003.
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