Caerulein-induced acute pancreatitis inhibits protein synthesis through effects on eIF2B and eIF4F

Maria Dolors Sans,1 Matthew J. DiMagno,2 Louis G. D'Alecy,1 and John A. Williams1,2

1Departments of Molecular and Integrative Physiology and 2Internal Medicine, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0622

Submitted 23 December 2002 ; accepted in final form 19 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Acute pancreatitis (AP) has been shown in some studies to inhibit total protein synthesis in the pancreas, whereas in other studies, protein synthesis was not affected. Previous in vitro work has shown that high concentrations of cholecystokinin both inhibit protein synthesis and inhibit the activity of the guanine nucleotide exchange factor eukaryotic initiation factor (eIF)2B by increasing the phosphorylation of eIF2{alpha}. We therefore evaluated in C57BL/6 mice the effects of caerulein-induced AP on pancreatic protein synthesis, eIF2B activity and other protein translation regulatory mechanisms. Repetitive hourly injections of caerulein were administered at 50 µg/kg ip. Pancreatic protein synthesis was reduced 10 min after the initial caerulein administration and was further inhibited after three and five hourly injections. Caerulein inhibited the two major regulatory points of translation initiation: the activity of the guanine nucleotide exchange factor eIF2B (with an increase of eIF2{alpha} phosphorylation) and the formation of the eIF4F complex due, in part, to degradation of eIF4G. This inhibition was not accounted for by changes in the upstream stimulatory pathway, because caerulein activated Akt as well as phosphorylating the downstream effectors of mTOR, 4E-BP1, and ribosomal protein S6. Caerulein also decreased the phosphorylation of the eukaryotic elongation factor 2, implying that this translation factor was not inhibited in AP. Thus the inhibition of pancreatic protein synthesis in this model of AP most likely results from the inhibition of translation initiation as a result of increased eIF2{alpha} phosphorylation, reduction of eIF2B activity, and the inhibition of eIF4F complex formation.

protein translation; mice


IT HAS BEEN WELL ESTABLISHED that experimental acute pancreatitis (AP) inhibits pancreatic exocrine secretion (32, 36, 55, 60). It has also been reported that pancreatic protein synthesis is unaffected by AP, leading to accumulation of digestive enzymes (5, 30, 55, 60). However, other studies have shown inhibition of pancreatic protein synthesis in both in vivo experimental AP studies (17, 26, 32, 36, 46) and in vitro models of AP using CCK hyperstimulation of isolated acini (31, 48, 56). This study seeks to clarify how protein synthesis is affected in an in vivo caerulein hyperstimulation model of AP in mice, using the "flooding dose" technique to measure protein synthesis and by analyzing the state of key regulatory mechanisms in protein translation.

Protein synthesis or translation of mRNA into protein has several known major regulatory steps involving initiation and elongation phases. The first major step in translation initiation is the binding of the initiator tRNAiMet to the 40 S ribosomal subunit, which is regulated by the eukaryotic initiation factor (eIF)2 (27, 51). In order for eIF2 to promote each round of initiation, GTP must replace bound GDP, a reaction catalyzed by the guanine nucleotide exchange factor eIF2B (27). eIF2B activity can be stimulated in various cells by hormones and trophic factors (51) that stimulate the phosphorylation of its {epsilon}-subunit through the phosphatidylinositide-3 kinase (PI3-kinase)/glycogen synthase kinase-3 (GSK-3) pathway (22, 62, 63). Inhibition of eIF2 occurs in various forms of cellular stress by phosphorylation of Ser51 in eIF2{alpha}, which converts the eIF2 from a substrate to a competitive inhibitor of eIF2B (58). It has been demonstrated in vitro that the inhibition of the eIF2B activity by phosphorylation of eIF2{alpha} is at least partially responsible for the inhibition of acinar protein synthesis at high CCK concentrations (56).

The second major regulatory step of translational control involves the formation of the eIF4F complex, which contains the mRNA cap-binding protein eIF4E along with initiation factors eIF4G and eIF4A. Assembly of this complex can be regulated by the availability of eIF4E, which is sequestered by eIF4E-binding proteins (4E-BPs). Phosphorylation of eIF4E-BP by several kinases, including the mammalian target of rapamycin (mTOR), releases eIF4E (18). In rat pancreas, enhancement of mRNA translation is believed to be mediated by the PI3-kinase-Akt-mTOR pathway (65). Physiological concentrations of CCK enhance phosphorylation of the binding protein 4E-BP1 (also known as PHAS-I), which leads to the release of eIF4E and the formation of eIF4F (7, 8). The PI3-kinase-Akt-mTOR pathway also activates another regulatory site in translation initiation, the 70-kDa ribosomal protein S6 kinase (p70S6K or S6K) (9, 39, 40). Activation of S6K induces phosphorylation of the ribosomal protein S6, which is believed to enhance mRNA translation for species with a terminal polypyrimidine tract (39). S6 phosphorylation usually occurs concomitant with an increase in protein synthesis (40). CCK activates the 70-kDa ribosomal protein S6 kinase in isolated acini through a wortmannin and rapamycin-sensitive pathway (6).

After translation initiation, amino acids are added sequentially to the growing peptide by the process of translation elongation. Elongation rates are also modulated by phosphorylation, particularly that of eukaryotic elongation factor 2 (eEF2) (44, 50). To be active, eEF2 must be dephosphorylated, whereas phosphorylation at Thr56 causes inactivation (50). Thr56 is phosphorylated by a specific, Ca2+/CaM-dependent eEF2 kinase, also known as calcium/calmodulin-dependent protein kinase III (53). Because activation of this kinase correlates with increases in intracellular Ca2+, phosphorylation of eEF2 could mediate inhibition of protein synthesis by high concentrations of CCK or caerulein (41).

The present study was designed to evaluate the extent of pancreatic protein synthesis inhibition during caerulein-induced AP and to investigate the responsible regulatory mechanisms. After it was established that inhibition of protein synthesis occurs during AP, we studied how this inhibition might be explained by changes in the translational machinery, including eIF2B activity, eIF4F complex formation, and eEF2 phosphorylation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Materials. Caerulein and bombesin were obtained from Bachem (Torrance, CA); JMV-180 was from Research Plus (Bayonne, NJ); GDP, DTT, and phenylalanine were from Sigma (St. Louis, MO). Goat anti-rabbit, anti-mouse IgG conjugated to horseradish peroxidase and enhanced chemiluminescent (ECL) reagent and L-[2,3,4,5,6-3H]phenylalanine were from Amersham Pharmacia Biotech (Piscataway, NJ); 10, 15, and 4-20% Tris · HCl precast gels and broad-range prestained SDS-PAGE standard markers were from Bio-Rad (Hercules, CA); nitrocellulose membranes were from Schleicher & Schuell (Keene, NH). [3H]GDP (11.3 Ci/mmol) was from New England Nuclear Life Science Products (Boston, MA); scintillation liquids Bio-Safe II and Filtron-X were from Research Products International (Mount Prospect, IL) and National Diagnostics (Atlanta, GA), respectively; 25-mm nitrocellulose filter discs (HAWP) and Waters AccQ · Fluor Reagent Kit were from Millipore (Milford, MA). Protein A-linked Sepharose beads were obtained from Pierce Chemical (Rockford, IL); GSK-3 monoclonal antibody was from Upstate Biotechnology (Lake Placid, NY); phospho-GSK-3{alpha}/{beta} (Ser21/9) polyclonal antibody, polyclonal eEF2, and phospho-eEF2 (Thr56) antibodies were from Cell Signaling (Beverly, MA); eIF4E-BP (PHAS-I) polyclonal antibody was from Calbiochem (San Diego, CA); and phospho-eIF2{alpha} (Ser51) polyclonal antibody was from Research Genetics, (Huntsville, AL). An eIF2{alpha} monoclonal antibody, originally developed by Dr. E. C. Henshaw, that recognizes both phosphorylated and unphosphorylated forms of eIF2{alpha} was also used. Polyclonal anti-phospho-eIF2B{epsilon} (Ser539) antibody was from Biosource International (Camarillo, CA). Mouse anti-eIF4E antibody was a gift from Dr. S. R. Kimball (Pennsylvania State University, Hershey, PA); and rabbit anti-eIF4G antibody was a gift from Dr. R. E. Rhoads (Louisiana State University, Shreveport, LA).

Induction of acute pancreatitis. AP was induced in mice by administering multiple supramaximal doses of the cholecystokinin analog caerulein (45). Adult male C57BL/6J mice, 6-10 wk old (6-10 per group) received hourly intraperitoneal injections of caerulein (50 µg/kg). All pancreatic parameters were analyzed at 10 min, 30 min, or 1 h after a single caerulein injection or 1 h after the last of three or five hourly injections. AP was confirmed by increased pancreatic trypsin activity, increased serum amylase or lipase levels, increased pancreatic weight used as a marker of edema, and by cytoskeletal disruption (14). Control studies with different secretagogues were also performed, administering caerulein at a low dose and high doses of the CCK analog JMV-180 and bombesin. Mice for these groups received three hourly intraperitoneal injections of caerulein at 0.5 µg/kg, JMV-180 at 5 mg/kg, and bombesin at 50 µg/kg. All animals were euthanized by CO2 asphyxiation and decapitation, and the pancreas was removed immediately. To measure the rate of protein synthesis and other biochemical parameters, the pancreas was either frozen in liquid N2 and stored at -80°C or homogenized with a Polytron homogenizer for immediate assays.

Protein concentration of the homogenates was assayed by use of the Bio-Rad protein assay reagent with BSA as standard. DNA concentration was measured by fluorescence assay using the fluorescent dye bisBENZIMIDE (Hoechst 33258), using a DNA quantitation kit from Sigma (St. Louis, MO).

Measurement of pancreatic protein synthesis. Pancreatic protein synthesis was determined using the flooding dose technique, originally described by Garlick and co-workers (16) and later validated by Sweiry and co-workers (61) in rat pancreas. Following the protocol of Lundholm and co-workers (33) to measure liver protein synthesis in mice, we injected 0.4 µCi/g of L-[3H]phenylalanine together with unlabeled L-phenylalanine (1.5 µmol/g) by the intraperitoneal route in a volume of 300 µl. Ten minutes later, blood was drawn by cardiac puncture into heparinized syringes, and pancreases were rapidly removed and frozen in liquid nitrogen. Frozen pancreas was subsequently homogenized in 10 vol of 0.6 N perchloric acid (PCA), whereas heparinized blood was also mixed (1:1) with 1.2 N PCA. In both cases, after 1 h of precipitation, samples were centrifuged at 10,000 g for 15 min and the supernatants were removed and neutralized with KOH before analysis for 3H and L-phenylalanine. In the pancreas samples, the original PCA precipitate was washed three times with 0.6 N PCA and resuspended in 0.3 M NaOH for determination of radioactivity and protein concentration. L-Phenylalanine was measured by HPLC on a C18 reverse phase column after precolumn derivatization with Waters AccQ-Fluor Reagent Kit to produce a stable fluorescent derivative of L-phenylalanine. Protein synthesis was calculated from the rate of radioactive L-phenylalanine incorporation into pancreatic protein using the specific radioactivity of pancreatic PCA-soluble L-phenylalanine as representative of the precursor pool and expressed as nanomoles of L-phenylalanine per milligram of protein.

In preliminary studies, we validated this method on ICR mice, and we found that the uptake of L-phenylalanine into the pancreas and its specific activity in the PCA soluble pool was maintained nearly constant from 5-20 min after the injection of the radioactive tracer. We chose 10 min as the optimal time to determine protein synthesis, because incorporation of 3H into protein was increasing linearly at this time point (M. D. Sans and J. A. Williams, unpublished results).

Measurement of eIF2B activity and phosphorylation state. Determination of eIF2B activity in pancreatic tissue was performed as described previously by measuring the rate of exchange of [3H]GDP present in an exogenous eIF2 · [3H]GDP complex for free nonradiolabeled GDP in pancreatic tissue samples (28, 56). The guanine nucleotide exchange activity was measured as a decrease in eIF2 · [3H]GDP complex bound to nitrocellulose filters and expressed as nanomoles of GDP exchanged per minute per milligram of acinar protein or as a percentage of the control group (56).

Another determinant of eIF2B activity is the phosphorylation state of its {epsilon}-subunit, which can be phosphorylated (and inhibited) by GSK-3. Pancreatic samples in SDS buffer were resolved in a 10% SDS-PAGE gel followed by Western blot analysis using an anti-phospho eIF2B{epsilon} (Ser539) polyclonal antibody (1:1,000) and detected by ECL.

Coimmunoprecipitation of eIF4G and eIF4E and formation of the eIF4F complex. To quantify the formation of the eIF4F complex, we analyzed the association of eIF4E with eIF4G by coimmunoprecipitation, as previously described (8). Briefly, pancreatic samples were homogenized in 2 ml of buffer, centrifuged at 10,000 g for 10 min at 4°C, and the supernatant, containing microsomes and soluble protein, was used to analyze translation factors. The association of eIF4G and eIF4E was assessed by analyzing the amount of eIF4G bound to eIF4E immunoprecipitated using specific anti-eIF4E antibody, following the protocol described by Kimball and coworkers (29). The immunoprecipitates were resolved on 4-20% gradient gel SDS-PAGE followed by Western blot analysis using anti-eIF4G antibody (1:2,000).

Measurement of Akt activity. Akt (protein kinase B) activity was measured using the nonradioactive Akt kinase assay kit from Cell Signaling (Beverly, MA). Briefly, pancreas samples were homogenized in 2 ml of lysis buffer containing (in mM): 20 Tris (pH 7.5), 150 sodium chloride, 1 EDTA, 1 EGTA, 2.5 sodium pyrophosphate, 1 {beta}-glycerophosphate, 1 sodium vanadate, and 1 PMSF, with 1% Triton X-100, 10 µg/ml leupeptin, and aprotinin. The homogenates were contrifuged for 15 min at 10,000 g and 4°C, and the supernatants were used to measure protein concentration and immunoprecipitate Akt. The resulting immunoprecipitate was then incubated with GSK-3 fusion protein as substrate at 30°C for 30 min in the presence of ATP and kinase buffer. Phosphorylation of GSK-3 was measured by Western blotting using an anti-phospho-GSK-3 {alpha}/{beta} (Ser21/9) antibody. Quantitation in this case and in all Western blot analyses was performed using Multi-Analyst software (Bio-Rad).

Evaluation of the phosphorylation state of 4E-BP1. The phosphorylation state of the 4E-BP1 was determined by protein immunoblot analysis using an antibody that recognizes all forms of 4E-BP1. 4E-BP1 resolves into multiple electrophoretic forms during SDS-PAGE depending on which, and how many, sites are phosphorylated (43). Unlike the more rapidly migrating forms ({alpha} and {beta}), the slowly migrating {gamma}-form does not bind to eIF4E. For this analysis, aliquots of pancreas lysates were boiled for 10 min, cooled to room temperature, and centrifuged at 1,000 g for 30 min at 4°C. Supernatant proteins were resolved in a 15% SDS-PAGE gel, transferred to nitrocellulose, and analyzed by Western blotting using the 4E-BP1 polyclonal antibody (1: 3,000) and ECL detection kit. The amount in the {gamma}-band was calculated as a percent of total.

Evaluation of the phosphorylation state of the eIF2{alpha}, eEF2, and the ribosomal protein S6. The phosphorylation state of these proteins was determined by the relative amount of eIF2{alpha}, eEF2, and S6 in the phosphorylated form, quantitated by protein immunoblot analysis using affinity-purified antibody that specifically recognizes eIF2{alpha} phosphorylated at Ser51, eEF2 phosphorylated at Thr56, and S6 phosphorylated at Ser240/244. For all analysis, aliquots of pancreas lysates were resolved in a 10% SDS-PAGE gel, transferred to nitrocellulose, followed by Western blot analysis using the anti-phospho eIF2{alpha} antibody (1:1,500), anti-phospho eEF2 antibody (1:1,000), and the anti-phospho S6 antibody (1:1,000) and detected by ECL. To ensure equal loading, the same membranes were stripped and reprobed for total eIF2{alpha} using a monoclonal antibody to eIF2{alpha}, diluted 1:500, total eEF2 using a polyclonal antibody to eEF2 at 1:1,000, and an S6 polyclonal antibody (1:500) for the ribosomal protein S6.

Statistical analysis. Data represent means ± SE and were obtained from 6-10 animals per group. Statistical analysis was carried out by one-way ANOVA, as calculated by the StatView program (SAS Institute Cary, NC). Differences with P < 0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Inhibition of pancreatic protein synthesis accompanies caerulein-induced AP. To study the effect of AP on pancreatic protein synthesis, we evaluated the level of L-[3H]phenylalanine incorporation into PCA-precipitable protein over a standard 10-min period at different time points after caerulein administration. Protein synthesis was inhibited 25% as early as 10 min after a single injection of caerulein (Fig. 1A). This inhibition was maintained during the first hour and further enhanced (~75% reduction) after three and five hourly injections of caerulein (Fig. 1A). The reduction in protein synthesis was not related to a decrease of the L-phenylalanine specific activity in the soluble fraction of the pancreas, because this parameter remained constant at all the time points (Fig. 1B). In studies of similar mice without administration of radioactivity, pancreatic trypsin activity showed an early peak at 30 min and serum amylase and lipase increased linearly over the 5 h (14). Because no statistical difference in rates of protein synthesis was observed among all control groups, which received saline injections, we analyzed the effects of caerulein on different parameters at the specified times by comparing them with the pooled control data from all time points.



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Fig. 1. Effect of caerulein-induced acute pancreatitis on L-[3H]phenylalanine incorporation into pancreatic protein in C57BL /6 mice (A) and specific activity of L-[3H]phenylalanine in the perchloric acid-soluble fraction of the pancreas (B). Caerulein was administered at 50 µg/kg ip, and protein synthesis was determined 10 min, 30 min, and 1 h after a single injection and 1 h after 3 and 5 hourly injections. Values are expressed as nanomoles of incorporated phenylalanine per 10 min per milligram of protein in A, and as dpm per nanomole L-phenylalanine in B, and are means and SE of 5 mice (saline groups) and 8-9 mice (caerulein groups). *P < 0.05 vs. control (saline groups).

 

As a control, we also measured the levels of total protein synthesis induced by other secretagogues. We administered three hourly injections of low-dose caerulein (0.5 µg/kg), high-dose bombesin (50 µg/kg), and high-dose JMV-180 [a low-affinity CCK-receptor agonist in mice (38)] at 5 mg/kg and measured L-[3H]phenylalanine incorporation into PCA-precipitable protein. Caerulein at this low dose induced an increase of ~50% in total protein synthesis after three hourly injections, compared with control animals (Table 1). JMV-180 at a dose 100 times higher than the high-dose caerulein (50 µg/kg) also had a small stimulatory effect on protein synthesis (Table 1). High doses of bombesin inhibited pancreatic protein synthesis, although not to as great an extent as caerulein (Table 1). This high dose of bombesin in mice is known to induce pancreatitis but to a lesser extent than high doses of caerulein (49). Thus inhibition of protein synthesis in vivo seems to accompany the cellular damage caused by the induction of AP.


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Table 1. Effect of various peptides on total pancreatic protein synthesis

 

Inhibition of translation initiation factor eIF2B activity accompanies caerulein-induced AP. After establishing that protein synthesis was inhibited in this model of AP, we examined the regulatory mechanisms of protein translation that could be involved in this inhibition. Because modulation of eIF2B activity is known to be one of the most important regulatory points in translation initiation (27), we studied the effect of caerulein on eIF2B activity at several time points. Extracts of control pancreas exhibited an eIF2B activity of 27.3 ± 2.9 nmol · min-1 · mg protein-1 (n = 15). eIF2B activity was lower 10 min after caerulein injection (22.8 ± 2.2 nmol · min-1 · mg protein-1), showed a significant reduction after 30 min (17.2 ± 1.9 nmol · min-1 · mg protein-1), and declined further after one and three hourly injections of caerulein to 40% of control (Fig. 2A).



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Fig. 2. Effect of caerulein-induced acute pancreatitis on eukaryotic initiation factor (eIF)2B activity (A), eIF2B{epsilon} phosphorylation (B), and eIF2{alpha} phosphorylation (C). The pancreas was removed 10 min, 30 min, and 1 h after a single-dose injection of caerulein at 50 µg/kg and 1 h after 3 hourly injections. Measurements of eIF2B activity and levels of phosphorylation of eIF2B{epsilon} and eIF2{alpha} are described in MATERIALS AND METHODS. The results are expressed as a percentage of pooled control levels. In B, the inset shows a representative blot for eIF2B{epsilon} phosphorylation levels, whereas in C, the insets show representative blots for eIF2{alpha} phosphorylation levels and total eIF2{alpha}. For A-C, data shown are the means and SE of 5-9 animals per group, *P < 0.05, #P < 0.01 vs. pooled control group.

 

To understand the cause of this reduced eIF2B activity, we first studied the phosphorylation of the {epsilon}-subunit of eIF2B, which is regulated by GSK-3. Second, the phosphorylation of eIF2{alpha} was assessed, because this is the best-characterized mechanism for inhibiting eIF2B activity and occurs by competing for GTP (27, 51). High doses of caerulein reduced the phosphorylation of eIF2B{epsilon} at all time points (Fig. 2B). This decrease in phosphorylation does not explain the reduced eIF2B activity, because eIF2B{epsilon} phosphorylation normally inhibits eIF2B activity (62). In contrast, inhibition of eIF2B activity can be explained by eIF2{alpha} phosphorylation, which was increased at all time points (Fig. 2C), reaching a maximum 30 min after a single injection of caerulein and persisting at the same level of phosphorylation after 1 h and three hourly injections (Fig. 2C). There was no change in total eIF2{alpha}. Combined, these results show that the inhibition of total protein synthesis in caerulein-induced AP is associated with the inhibition of eIF2B activity, and this inhibition, in turn, is most likely related to the increased phosphorylation of eIF2{alpha}.

Caerulein-induced AP inhibits the formation of the eIF4F complex. The formation of the eIF4F complex (one of the main regulatory points on translation initiation and downstream of 4E-BP1 phosphorylation) was also reduced following caerulein injection as assessed by the association of the mRNA cap binding protein eIF4E with eIF4G by coimmunoprecipitation. The formation of the eIF4F complex was dramatically reduced after 30 min and recovered somewhat but was still significantly reduced after 1 h and after three hourly injections (Fig. 3A). This inhibition was associated with and could be explained, at least in part, by the degradation of eIF4G seen in the coimmunoprecipitation samples. This finding was confirmed by changes in total eIF4G by Western blotting of cell lysates (Fig. 3B). Clear degradation of this protein was seen 30 min after caerulein administration and in some cases after 1 h but not at earlier or later time points. By contrast, there was no change in the total level of eIF4E (Fig. 3A). From these results, we conclude that a reduction of eIF4F complex formation may contribute to a reduction in total protein synthesis.



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Fig. 3. Effects of caerulein-induced acute pancreatitis on eIF4F complex formation, measured as eIF4E and eIF4G coimmunoprecipitation (A). Data for coimmunoprecipitated eIF4G are expressed as percentage of pooled control levels and SE for 6-8 animals per group. Representative blots for eIF4G associated with eIF4E and total eIF4E are shown in the insets. #P < 0.01 vs. pooled control group. B: representative Western blotting showing the degradation of total pancreatic eIF4G 30 min after caerulein injection.

 

Caerulein-induced AP activates the Akt/mTOR pathway. Because eIF4F (one of the downstream effectors of the Akt-mTOR pathway) was inhibited in the development of AP, it was important to know whether its upstream effectors were also inhibited. The Akt/mTOR pathway is believed to be activated by physiological doses of CCK in the pancreas (65), and our results show that it was also activated by supramaximal doses of caerulein in vivo (Figs. 4 and 5). Pancreatic Akt activity was rapidly increased threefold 10 min after a single caerulein injection and remained significantly elevated at later times after a single or three hourly injections (Fig. 4). Two of the downstream effector molecules of mTOR, eIF4E-binding protein and the ribosomal protein S6, were also activated (phosphorylated) after caerulein hyperstimulation. Similarly to the Akt activity, the phosphorylation of the eIF4E-BP1 was increased at all time points and was significantly enhanced from 30 min after a single caerulein injection until the third hourly injection, with a maximum after 1 h (Fig. 5A). The phosphorylation of the ribosomal protein S6 was only significantly increased after one and three hourly injections (Fig. 5B). From these results, we conclude that the Akt/mTOR pathway, which is a known stimulatory pathway for pancreatic protein synthesis, is activated by a supramaximal dose of caerulein to the 4E-BP1 and S6K level. Whereas Akt may well have other effects on cell survival (9), its effects to activate protein synthesis, however, are blocked by the decrease in the eIF4F complex formation.



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Fig. 4. Effect of caerulein-induced acute pancreatitis on Akt activity 10 min, 30 min, and 1 h after a single injection of caerulein at 50 µg/kg and 1 h after 3 hourly injections. Activity is expressed as percentage of pooled control levels, and each columm is the mean and SE of 6-9 animals per group. A representative blot for GSK3 substrate phosphorylation levels is shown. *P < 0.05, #P < 0.01 vs. pooled control group.

 


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Fig. 5. Effect of caerulein-induced pancreatitis on the phosphorylation of 2 of the downstream targets of the mTOR pathway: the eIF4E binding protein, 4E-BP1 (A) and the ribosomal protein S6 (B). A: the phosphorylation levels of 4E-BP1 as the amount of 4E-BP1 in the {gamma}-phosphorylated form as a percentage of the total 4E-BP1. Inset: representative immunoblot with positions of {alpha}-, {beta}-, and {gamma}-forms of 4E-BP1 are noted on the left. The most highly phosphorylated {gamma}-form, which exhibits the slowest electrophoretic mobility, does not bind eIF4E. B: the phosphorylation levels of S6 as percentage of pooled control levels. Representative blots for S6 phosphorylation and total S6 are shown in the insets. For both panels, values are means and SE of 6-9 animals per group, *P < 0.05, #P < 0.01 vs. pooled control group.

 

Caerulein-induced AP does not cause general protein degradation. Because eIF4G degradation was observed 30 min after a single caerulein injection and occurred concurrently with an early maximal increase in pancreatic trypsin activity (14), we determined whether there could be general protein degradation in our model of AP. Of the specific proteins evaluated, ribosomal protein p70 S6 kinase (S6K) was also partially degraded at this time point but recovered to control levels after 1 h of caerulein administration (data not shown). However, no change in total protein concentration (related to total DNA) was seen at any of the studied time points (Table 2). Moreover, we failed to see evidence of protein degradation in SDS-PAGE gels stained with coomassie blue, because no differences in the protein-ladder pattern were observed between the caerulein-treated groups and their controls (Fig. 6). From these results, we conclude that the limited proteolysis or protein degradation occurring during the early course of AP is a specific rather than a global effect, because there is no general increase in protein degradation at the time points analyzed in this study.


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Table 2. Total protein concentration (mg/µg DNA) in pancreas with caerulein-induced acute pancreatitis at different time points

 


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Fig. 6. Effects of caerulein-induced acute pancreatitis on total protein showing the absence of general protein degradation. Representative image of a Coomassie blue-stained gel of pancreatic homogenates at different time points after caerulein injection. MW, molecular weight.

 

Caerulein-induced AP does not inhibit eEF2. Finally, we tested whether phosphorylation of eEF2 could be related to the inhibition of total protein synthesis seen in our model, because peptide elongation is another potential regulatory step for protein synthesis, and it is known that a Ca2+/CaM-activated kinase phosphorylates Thr56 and inactivates eEF2 (53). Our results, however, showed a reduction in phosphorylation of Thr56 in this elongation factor 10 and 30 min after caerulein administration, which would be expected to increase its activity (Fig. 7). There was a return toward a basal phosphorylated state of eEF2 after one and three hourly injections. There was no change in the amount of total eEF2 at any time point (Fig. 7). These results indicate that eEF2 activity is not inhibited and therefore will not mediate the inhibition of protein synthesis seen following caerulein-induced AP.



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Fig. 7. Effects of caerulein-induced acute pancreatitis on the phosphorylation state of eukaryotic elongation factor 2 (eEF2) 10 min, 30 min, and 1 h after a single injection of caerulein and 1 h after 3 hourly injections. Phosphorylation levels of eEF2 are shown as percentage of pooled control levels and are the means and SE of 6-9 animals per group. Representative blots for eEF2 phosphorylation and total eEF2 are shown in the insets. #P < 0.01 vs. pooled control group.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The present work was designed to clarify the effect of caerulein-induced AP on pancreatic protein synthesis and, more importantly, to provide insight of how caerulein-induced AP affects the regulation of protein translation in mice. Global pancreatic protein synthesis, which primarily results in the production of digestive enzymes and proenzymes, was progressively inhibited as early as 10-30 min after high-dose caerulein administration, concurrent with the appearance of other early features of AP. The inhibition of protein synthesis correlated with a reduction in eIF2B activity due to eIF2{alpha} phosphorylation and a reduction in the eIF4F complex formation due, in part, to eIF4G degradation. Inhibition of eIF4F formation occurred despite the fact that the Akt-mTOR pathway, which physiologically stimulates translation after a meal, was activated. eEF2, the key regulator of translation elongation, was not inhibited (phosphorylated) by the onset of AP.

Well-characterized disease models of experimental AP share several common features, including secretory blockade (32, 36, 60), intracellular trypsin activation (20, 55, 60), high levels of digestive enzymes in blood, cytoplasmic vacuolization (36, 60), and activation of NF-{kappa}B with later induction of an inflammatory response (11). The effect of AP on pancreatic protein synthesis observed in these and other studies has been less clear, because some studies reported no effect (5, 30, 55), whereas other studies reported inhibition (17, 26, 32, 36). In the present study, AP was induced in C57BL/6 mice using a caerulein hyperstimulation model involving single or repetitive doses of caerulein (14, 45). The development of AP was associated with progressive inhibition of protein synthesis beginning as early as 10 min after the first caerulein injection and reaching a maximum inhibition after three hourly injections. Similar inhibitory effects on pancreatic protein synthesis have also been reported using a caerulein hyperstimulation model in rats (32) as well as other more severe models of AP, including a taurocholate model in pigeons and rats (36) and choline-deficient, ethionine-supplemented (CDE) diet in mice (17).

Different methods have been used to measure protein synthesis involving administration of labeled amino acids with subsequent measurement of the incorporation of label into protein. Accurate assessment of the specific radioactivity of the precursor amino acid at the site of protein synthesis is important. The use of readily accessible compartment pools, such as the intracellular or plasma pools, to estimate precursor labeling is based on the assumption that the experimental treatment does not alter the relationship between the labeling of the sampled pool and that of the aminoacyl-tRNA, the direct precursor of protein synthesis. However, some experimental conditions have the potential to alter precursor enrichment either by affecting the amount of labeled amino acid entering the cell or by affecting the contribution of unlabeled amino acids derived from protein degradation to the charging of aminoacyl-tRNA. In these cases, the problem of accurately determining precursor enrichment may be minimized by the flooding dose technique (10). With the flooding dose technique, the labeled amino acid is injected, not as a tracer, but contained in a large (i.e., much larger than the endogenous free amino acid pool) bolus of unlabeled amino acid, making the specific activities in all free amino acid compartments more alike than if the labeled compound is given as a tracer dose. Thus the labeling of aminoacyl-tRNA is less likely to be affected by experimental manipulations. In addition, the large amount of amino acid injected ensures that the specific activity in the free pools remains almost constant for a certain period of time after injection (13). We used L-[3H]phenylalanine as a radioactive tracer, because Sweiry and co-workers (61) demonstrated that phenylalanine transport across the baso-lateral membrane of the pancreatic acinar cells is not a rate-limiting factor for protein synthesis. In the present study, amino acid uptake was not altered by AP, because no change occurred in the pancreatic PCA-soluble L-phenylalanine specific activity at any of the studied time points (Fig. 1B). Thus the observed inhibition of pancreatic protein synthesis is not likely related to a decrease in the pancreas precursor pool uptake. Finally, the flooding dose technique is advantageous in the present study not only because it is reliable, but also because it can be used in unrestrained and unanesthetized animals (13) and has been well validated in muscle (10, 16), liver (33), and pancreas (61).

In control studies measuring total protein synthesis, we used a low dose of caerulein designed to mimic physiological stimulation of secretion; stimulation of protein synthesis was observed similar to the effect of physiological stimulation of rat acini by CCK in vitro (31, 56). We also studied the effects of the CCK receptor agonist JMV-180 (38), because in rats, JMV-180 acts as a partial agonist that stimulates secretion but fails to induce pancreatitis. We used a 5-mg/kg dose, which was 100 times the high dose of caerulein and the highest amount we could solubilize and deliver readily to mice. At this dose, JMV-180 slightly stimulated protein synthesis. In the mouse, however, JMV-180 is a full agonist, and it is likely that if a higher dose could have been given, it might have inhibited protein synthesis as it does in vitro (24, 38). We studied high doses of bombesin because the mechanism of action of bombesin on acinar cells appears to be similar in many, but different in some, respects compared with CCK (15). In the mouse, high doses of bombesin (50 µg · kg-1 · h-1)in vivo have been reported to induce pancreatic edema, increase leukocyte infiltration, and increase necrosis similar to but to a lesser extent than does the same dose of caerulein (49). Moreover, in vitro, high concentrations of bombesin inhibit amylase secretion (4). This contrasts to the rat, in which high concentrations of bombesin do not induce AP (66) and do not inhibit protein synthesis or amylase secretion in rat pancreatic acini in vitro (56). Thus, in the mouse, inhibition of protein synthesis correlates with the extent of pancreatitis. Further work is necessary to evaluate these interesting species diferences.

To understand how protein synthesis was inhibited by caerulein-induced AP, we determined which initiation factors could be involved in this model of cell stress. In AP, which is associated with secretory blockade and the autoactivation of digestive enzymes (32, 55), other stress-inducing factors such as a decrease of intracellular ATP levels (34, 35), increase of free radicals (12, 57), or increase of cytoplasmic calcium (47, 69) may be involved. In nonpancreatic cell types, similar stress situations such as ischemia (64), perturbation in cellular calcium (25), and apoptosis (54) inhibit protein synthesis in a manner similar to the endoplasmic reticulum (ER) stress response (52). It is quite likely that in our study, the reduction of total protein synthesis is due to an ER stress response induced by a decrease of intracellular ATP levels, an increase of free radicals, an increase of cytoplasmic calcium, or all of these at the same time. In these conditions, inhibition of protein synthesis is often accompanied by an increase in the phosphorylation of the {alpha}-subunit of eIF2, which leads to a reduction in the activity of eIF2B (1, 27). The phosphorylation of eIF2{alpha} is known to be brought about by activation of the ER resident kinase PERK, which possesses a luminal domain sensitive to stress and a cytoplasmic kinase domain specific for eIF2{alpha}. Because levels of eIF2 in cells are two- to fivefold higher than that of eIF2B, partial phosphorylation of eIF2{alpha} is sufficient to inhibit all of the eIF2B and to prevent the recycling of eIF2 (25, 27). A reduction in eIF2B activity has been observed in pancreas, in isolated acini, in response to high concentrations of CCK where depletion of ER calcium stores initiates ER stress and increases eIF2{alpha} phosphorylation (56). In the present in vivo study, reduced eIF2B activity was seen starting 30 min after caerulein administration and could be due to either or both depletion of ER calcium in response to caerulein or the cellular stress that accompanies AP. Interestingly, this occurs despite a significant reduction in eIF2B{epsilon} phosphorylation at the GSK-3 phosphorylation site (Fig. 2B), which ordinarily activates eIF2B but is ineffective in the setting of an eIF2B inhibition by eIF2{alpha}. The molar ratio of eIF2 to eIF2B in pancreatic acini is 3:1 (56), and because eIF2{alpha}(P) inhibits eIF2B activity on an approximately equimolar basis (27), a small increase in eIF2{alpha} phosphorylation would be sufficient to block eIF2B activity regardless of the potential activation that would result from a reduction in eIF2B{epsilon} phosphorylation.

We also found that the induction of AP inhibited the formation of the eIF4F complex, another key regulated step in regulation of translation. The association of eIF4E with eIF4G to form the eIF4F complex was almost completely blocked after 30 min of caerulein administration, a time at which there was only partial inhibition of protein synthesis. This clearly shows that inhibition of the eIF4F complex is not the sole mechanism responsible for inhibition of protein synthesis. In addition, eIF4F may not be necessary after initiation of translation due to formation of a cyclic initiation complex involving the poly A binding protein. The inhibition of eIF2B occurs more slowly and may be more important in the inhibition of protein synthesis. The degradation of eIF4G observed in this study after 30 min of caerulein administration could account for the inhibition of the eIF4F complex formation at this time point. We saw a decline in eIF4G degradation 1 h after one caerulein injection and a restitution of eIF4G levels 1 h after three hourly injections, possibly indicating an attempt to restore protein synthetic pathways; however, impaired binding of eIF4E with eIF4G persisted. Thus, whereas the inhibition of the eIF4F complex at 30 min can be completely explained by eIF4G degradation, the inhibition observed at later time points must be attributed to other mechanisms. Because 4E-BP1 phosphorylation is increased at all time points, an increase in the binding of eIF4E cannot be evoked as a cause of the inhibition of eIF4F complex formation. Degradation of the other components of the eIF4F complex is unlikely to account for this inhibition because we did not find degradation in the total amounts of eIF4E (Fig. 3A).

This selective, time-dependent degradation of eIF4G (and possibly also the ribosomal protein S6 kinase) might be explained by comparison with other cell types. It is known that in cells infected with picorna-viruses, the integrity of eIF4G declines after infection due to cleavage of eIF4G by viral and/or cellular proteases (18, 68). This cleavage destroys the bridging function of eIF4G in protein synthesis and leads to the inhibition of cap-dependent initiation (18). eIF4G can also be cleaved by caspase-3 (37) and -8 (42) in apoptotic cells. Pancreatic eIF4G could be cleaved by these caspases because caspase-3, -8, and -9 have all been reported to be activated in AP (3, 19). Alternatively, degradation of eIF4G could also involve protease activation independent of caspases, because in the model used, pancreatic trypsin activity is maximal after 30 min of caerulein administration (14). However, protein degradation occurring during AP in this study appeared to be a selective rather than a global effect, because total amounts of most of the initiation factors analyzed were not affected and general protein degradation was not observed in our samples from the AP group compared with control (Table 2 and Fig. 6).

It has been established that newly synthesized digestive enzymes accumulate within the pancreas during the course of AP (60). Some studies (17) have described an increase in total protein content during the development of an experimental edematous AP because of the blockade of secretion. In our study, total protein concentration was not modified at any time point during the development of AP, compared with the control animals (Table 2). These results could be explained by a balance established between an increase of the intracellular content of zymogens due to the secretory blockade, the inhibition of protein synthesis, and the loss of some zymogens into the extracellular space during the course of AP. Changes in gene expression could also account for an effect on translation rates that would lead to changes in specific proteins. It has been described that gene expression is altered during AP in an unparallel and noncoordinated manner (21, 67), and thus, whereas the expression of some proteins might be reduced, the expression of others would be increased. These effects taken together would be expected to increase over time and may well lead to a latter decrease in total pancreatic protein.

Caerulein-induced AP was accompanied by activation of the main known stimulatory pathway of acinar cell protein synthesis, the Akt-mTOR pathway. The stress activation of Akt is also not surprising, because it has been demonstrated that other types of cell stress can activate Akt (59). It can also be viewed as a result of overstimulation of CCK receptors in this model of pancreatitis. Two of the downstream effectors of Akt, 4E-BP1 and the ribosomal protein S6, were also activated (phosphorylated). However, the usual stimulatory effect on 4E-BP1 phosphorylation observed under physiological stimulation did not lead, in this case, to an increase in the formation of the eIF4F complex (as discussed above). In the case of S6, the lack of phosphorylation at earlier time points may be explained because its kinase (S6K) appeared to be partially degraded at 30 min, as mentioned earlier. However, there was a dramatic increase in S6 phosphorylation after one and three hourly injections that correlated, in turn, with the restitution of total eIF4G protein. Because the ribosomal protein S6 is implicated in the translation of a specific set of mRNAs that often encode proteins involved in the process of translation, such as ribosomal proteins and elongation factors (40), it is conceivable that S6 phosphorylation might facilitate the synthesis of a subclass of proteins including the translational proteins eIF4G and S6K. Thus the activation of the Akt-mTOR pathway might indicate that although protein synthesis is inhibited, some translation machinery is "primed" for resumption of normal protein synthesis.

Finally, caerulein-induced cell stress does not appear to inhibit translation factor eEF2. eEF2, a key regulated component in peptide chain elongation, can be regulated both through the Akt-mTOR pathway and by a Ca2+-activated kinase. Whereas the Akt-mTOR pathway activates eEF2 by activating a phosphatase that dephosphorylates eEF2 (50), CaM-kinase III inhibits eEF2 activity by phosphorylating the Thr56 residue, apparently by inhibiting its ability to interact with the ribosome (50). Our results showed a decrease in eEF2 phosphorylation 10 and 30 min after caerulein administration; providing evidence that eEF2 activity was not likely reduced and did not contribute to inhibition of protein synthesis at these time points. At the later time points, eEF2 phosphorylation levels returned close to the control level. It is possible that this time course reflects the sum of competing processes, but in any case, the net effect will not be inhibitory.

In summary, we conclude that the caerulein hyperstimulation model of AP in C57BL/6 mice inhibited protein synthesis through the inhibition of several regulatory mechanisms of protein translation, primarily, the guanine nucleotide exchange factor (eIF2B) and secondly, through the inhibition of the eIF4F complex formation. The inhibition of pancreatic protein synthesis in caerulein-induced AP most likely represents a protective response to cell stress, as observed in nonpancreatic cell types (23, 25, 64). Because ATP is necessary to maintain the protein synthesis mechanisms in the pancreas (2) and because it has been described that ATP levels in AP are diminished in mouse (35) and rat models (34, 35), the inhibition of the metabolically demanding process of protein synthesis may allow for the use of the cell's energy for other critical cell-saving functions to prevent further cell damage. Because this study has focused on a model of AP induced by caerulein hyperstimulation, further work is needed to determine whether the changes observed in this model apply to other models of AP.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This research was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (Grants DK-52860 and DK-59578) and by the Michigan Gastrointestinal Peptide Center (DK-34933).


    ACKNOWLEDGMENTS
 
We thank S. Kimball of Pennsylvania State Medical School for generous provision of reagents and ongoing advice on translational control, C. Logsdon of Univ. of Michigan for helpful advice during the development of the study and preparation of the present manuscript, and Q. Xie and S. E. Whitesall for technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. D. Sans, 1301 E. Catherine St., 7737 Med Sci II, Ann Arbor, MI 48109-0622 (E-mail: mdsansg{at}umich.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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