Feeding activates protein synthesis in mouse pancreas at the translational level without increase in mRNA
Maria Dolors Sans,1
Sae-Hong Lee,1
Louis G. D'Alecy,1 and
John A. Williams1,2
Departments of 1Molecular and Integrative Physiology and 2Internal Medicine, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0622
Submitted 3 December 2003
; accepted in final form 20 April 2004
 |
ABSTRACT
|
---|
To determine the mechanism of meal-regulated synthesis of pancreatic digestive enzymes, we studied the effect of fasting and refeeding on pancreatic protein synthesis, relative mRNA levels of digestive enzymes, and activation of the translational machinery. With the use of the flooding dose technique with L-[3H]phenylalanine, morning protein synthesis in the pancreas of Institute for Cancer Research mice fed ad libitum was 7.9 ± 0.3 nmol phenylalanine·10 min1·mg protein1. Prior fasting for 18 h reduced total protein synthesis to 70 ± 1.4% of this value. Refeeding for 2 h, during which the mice consumed 29% of their daily food intake, increased protein synthesis to 117.3 ± 4.9% of the control level. Pancreatic mRNA levels of amylase, lipases, trypsins, chymotrypsin, elastases, as well as those for several housekeeping genes tested were not significantly changed after refeeding compared with fasted mice. By contrast, the major translational control pathway involving Akt, mTOR, and S6K was strongly regulated by fasting and refeeding. Fasting for 18 h decreased phosphorylation of ribosomal protein S6 to almost undetectable levels, and refeeding highly increased it. The most highly phosphorylated form of the eIF4E binding protein (4E-BP1) made up the 14.6% of total 4E-BP1 in normally fed animals, was only 2.8% after fasting, and was increased to 21.4% after refeeding. This was correlated with an increase in the formation of the eIF4E-eIF4G complex after refeeding. By contrast, feeding did not affect eIF2B activity. Thus food intake stimulates pancreatic protein synthesis and translational effectors without increasing digestive enzyme mRNA levels.
digestive enzymes; protein translation; mice
PANCREATIC PROTEIN SYNTHESIS is required for production of secreted digestive enzymes, growth of the pancreas, and the replacement of normal cellular components. In the mature pancreas,
90% of protein synthesis has been estimated to be devoted to a mixture of about 20 digestive enzymes (34, 39). Digestive enzymes are secreted after food intake, with secretion mediated largely by neural and gastrointestinal hormone stimulation. To match digestive enzyme synthesis to dietary need, synthesis of digestive enzymes also needs to be controlled. Early studies (8, 46) showed that prolonged fasting decreased both pancreatic protein content and protein synthesis and that these were restored by refeeding. Whereas long-term dietary changes are known to affect mRNA expression (46), short-term meal-to-meal control needs to be immediate, reversible, and flexible. Such control of protein synthesis in other cell types is mainly at the translational level (25, 26).
Early studies on the neural and hormonal regulation of pancreatic protein synthesis were conflicting as reviewed by Case (8). With the advent of isolated acini and pancreatic lobules, gastrointestinal hormones, cholinergic stimulation, and insulin were all shown to stimulate protein synthesis (1921, 40). Rausch et al. (33) showed that in vivo infusion of CCK and secretin increased the rate of total protein synthesis and that of specific digestive enzymes measured subsequently in vitro in isolated lobules with little or no change in mRNA during the first 24 h of stimulation. The studies with isolated acini showed that the stimulatory effect was directly on acini and also did not require synthesis of new mRNA (1921, 31).
Recent studies (4, 5) have shown that pancreatic secretagogues can activate the translational machinery both in vitro and in vivo (6). The primary pathway being activated in acinar cells is the PI3K-PKB-mTOR pathway (38). In the rat pancreas, CCK stimulation leads to phosphorylation of eIF4E-BP1 (also known as PHAS-I), which leads to the release of eIF4E, the mRNA cap binding protein, and its subsequent incorporation into the eIF4F complex. In addition, CCK stimulation activates S6K, leading to the phosphorylation of ribosomal protein S6, which is believed to enhance translation of mRNA species with a terminal oligopyrimidine tract (26, 27). These effects are all blocked in vitro by rapamycin, an inhibitor of mTOR (5). The other major regulatory site in many cells, eIF2B, was not activated by CCK in vitro, although its inhibition may reduce acinar protein synthesis (35).
The present study was designed to evaluate whether meal- stimulated pancreatic protein synthesis is accompanied by translational activation, changes in mRNA levels, or both. We studied the effect of fasting and refeeding in mice to model meal-stimulated pancreatic secretion and digestive enzyme synthesis. After it was established that pancreatic protein synthesis was stimulated by normal refeeding, we analyzed the possible changes in the mRNA of several digestive enzymes and confirmed that the increase in protein synthesis was not due to the increase of mRNA but rather was accompanied by the activation of the translational machinery, specifically eIF4F complex formation and S6K activation. No change was seen in the activity of eIF2B.
 |
MATERIAL AND METHODS
|
---|
Materials.
GDP, DTT, phenylalanine, and SYBR Green were from Sigma (St. Louis, MO). TRIzol reagent and all oligonucleotides were from Invitrogen (Carlsbad, CA); RNeasy and the RNase-Free DNase set were from Qiagen (Valencia, CA); TaqMan (reverse transcription reagents), dNTPs and the Primer Express Primer software were from Applied Biosystems (Foster City, CA). Goat anti-rabbit and anti-mouse IgG conjugated to horseradish peroxidase and enhanced chemiluminescence (ECL) reagent were from Amersham Pharmacia Biotech (Piscataway, NJ); 10, 15, and 420% Tris·HCl precast gels, broad-range prestained SDS-PAGE standard markers, and fluorescein and the iCycler iQ real-time PCR detection system software 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); L-[2,3,4,5,6-3H]phenylalanine (L-[3H]Phe) was from Amersham Biosciences (Piscataway, NJ); the 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), and polyclonal phospho-GSK-3
/
(Ser21/9), Akt, and phospho-Akt (Ser473), ribosomal protein S6 kinase, ribosomal protein S6, and phospho-S6 (Ser240/244) antibodies were from Cell Signaling (Beverly, MA); eIF4E binding protein (PHAS-I) polyclonal antibody was from Calbiochem (San Diego, CA). Mouse anti-eIF4E antibody was a gift from Dr. Scot 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).
Experimental design.
All studies were carried out on male ICR mice (Harlan, Indianapolis, IN) weighing 2632 g fed Purina 5001 chow (LabDiet, St, Louis, MO) and placed in a 12:12-h light-dark cycle changing at 6:00 AM and 6:00 PM. Food intake was determined using powder chow in computer-monitored feeding cages using ChannelManager software (Stoelting Wood Dale, IL) and averaged per hour over a 72-h period. The average mouse consumed 5.5 ± 0.9 g food/day, with most consumed from 2:009:00 PM but with a second peak between 4:00 and 6:00 AM. When mice were provided water ad libitum but were fasted 18 h and then refed (at 9:00 AM), they consumed 1.6 ± 0.3 g or 29% of their normal daily intake in 2 h, with most in the first hour. For physiological studies, mice were fed pelleted chow and separated into different groups: fed (ad libitum), fasted (for 18 h, starting at 3:00 PM), refed 1, 2, or 3 h after the 18-h fast, and refasted 6 h (fasted for 18 h, then refed for 2 h followed by refasting for 6 h).
Measurement of pancreatic protein synthesis.
Pancreatic protein synthesis was determined using the flooding dose technique, originally described by Garlick et al. (12). With the use of the protocol of Lundholm et al. (24) used to measure liver protein synthesis in mice, we injected 0.4 µCi/g of L-[3H]Phe together with unlabeled L-Phe (1.5 µmol/g) by the intraperitoneal route in a volume of 300 µl. Ten minutes later, pancreases were rapidly removed and frozen in liquid nitrogen. Frozen pancreas was subsequently homogenized in 10 vol of 0.6 N perchloric acid (PCA) and processed as described previously (36). L-Phe 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. Protein synthesis was calculated from the rate of radioactive L-Phe incorporation into pancreatic protein using the specific radioactivity of pancreatic PCA-soluble L-Phe as representative of the precursor pool and expressed as nanomoles of L-Phe per milligram of protein.
RNA isolation, reverse transcription, and quantitative real time-PCR.
Total RNA was isolated from mouse pancreas using TRIzol and RNeasy spin columns. Purity and concentration of the isolated RNA were examined by OD280/OD260 ratios and by agarose gel electrophoresis and ethidium bromide staining. Total RNA (200 ng) was reverse transcribed using TaqMan reverse transcription reagents with random hexamers as primers and cDNA quantified in a spectrophotometer. Quantitative PCR was carried out using an I-Cycler IQ real-time PCR detection system for a 96-well plate from Bio-Rad. For each reaction, 10.3 µl HPLC water, 2 µl 10x PCR buffer, 2.2 µl MgCl2 (50 mM), 1 µl flourescein, 0.4 µl dNTP (10 mM), 2 µl primer mixtures [consisting of 0.1 nmol/µl concentration of each forward and reverse primers (Table 1) and 1 µl 100x SYBR Green in 100 µl volume], and 2 µl cDNA (0.5 µg/µl) were mixed before the run. PCR was performed in triplicate wells for each sample at 95°C for 3 min, 60°C for 1 min (repeated 40 times), and 55°C for 1 min. To distinguish between products and primer-dimers, we monitored the melt curve, obtained by increments of 0.5°C every 10-s interval from 55°C until it reached 96°C. Finally, all primer pairs were also validated with standard dilution curve (1:2, 1:4, 1:8, 1:16, 1:32). Data were analyzed using I-Cycler IQ real-time PCR detection system software to analyze the melt curve, the standard curve, and the quantitative amplification. The fluorescence resulting from the incorporation of SYBR Green 1 dye into the double-stranded DNA produced during the PCR reaction was quantitated to obtain the threshold cycle (CT) value for each sample. Because CT readings represent measurements on a log scale, a mean CT value was calculated for fed mice and the final relative amounts of mRNA for each animal were calculated as 2
CT, where
CT is the mean control CT minus the individual CT. This converts the log scale CT values to a relative number in linear form that can be used to calculate a mean ± SE for each group.
Gene sequences for housekeeping genes and digestive enzymes were primarily obtained from the GenBank NCBI Sequence Viewer (http://www.ncbi.nlm.nih.gov). The mouse pancreatic triglyceride lipase sequence was obtained by PCR amplification from reverse-transcribed mouse pancreatic RNA, using primers based on an EST sequence (AK002353
[GenBank]
) and the rat lipase sequence (M58369
[GenBank]
) and sequenced in both directions with multiple overlapping primers. The resulting full-length sequence is now present in GenBank as AY387690
[GenBank]
. Primers for quantitative RT-PCR (Table 1) were designed using the Primer Express software from ABI (Foster City, CA).
Measurement of PKB/Akt activity.
Activity of the Akt kinase was measured using the nonradioactive Akt kinase assay kit from Cell Signaling, as described previously (36). Briefly, pancreas samples were homogenized in 2 ml lysis buffer, homogenates were contrifuged for 15 min at 10,000 g and 4°C, and Akt was immunoprecipitated from 500 µg protein. 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 blot analysis using an anti-phospho-GSK-3
/
(Ser21/9) antibody. Quantitation in this case, and in all Western blot analysis, was performed using Multi-Analyst software (Bio-Rad).
Measurement of eIF2B activity.
Determination of eIF2B activity in pancreatic tissue was performed as described previously (17, 35) 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. 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 (35).
Evaluation of the phosphorylation state of Akt, the ribosomal protein S6, and eIF4E binding protein 4E-BP1.
The phosphorylation state of Akt and ribosomal protein S6 was determined by the relative amount of Akt or S6 in the phosphorylated form, quantitated by protein immunoblot analysis using affinity-purified antibody that specifically recognizes Akt and S6 when phosphorylated at Ser473 and Ser240/244, respectively. Aliquots of pancreas lysates were resolved in a 10% SDS-PAGE gel, transferred to nitrocellulose, followed by Western blot analysis using the anti-phospho Akt and S6 antibodies (1:1,000) and detected by ECL. To evaluate possible changes in the amount of these proteins, the same membranes were stripped and reprobed for total Akt (with an antibody that recognizes all forms) and ribosomal protein S6 using polyclonal antibodies to Akt and S6 at 1:500.
The phosphorylation state of the eIF4E-binding protein (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 (30). Unlike more rapidly migrating forms (
and
), the form exhibiting the slowest migration, referred to as the
-form, does not bind to eIF4E. For this analysis, aliquots of pancreas lysates were boiled for 10 min, and after they were cooled to room temperature, they were centrifuged at 1,000 g for 30 min at 4°C. Supernatant proteins were resolved in a 15% SDS-PAGE, transferred to nitrocellulose, followed by Western blot analysis using the 4E-BP1 polyclonal antibody (1:3,000), and detected by ECL. The amount in the
-band was calculated as a percentage of the total.
Coimmunoprecipitation of eIF4G with 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 (6). Briefly, pancreatic samples were homogenized in 2 ml 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, as described previously (36). The immunoprecipitates were resolved on 420% gradient SDS-PAGE followed by Western blot analysis using anti-eIF4G antibody (1:2,000).
Statistical analysis.
Data are reported as means ± SE, which were obtained from three to five different experiments with three to five animals per group in each experiment. Statistical analysis was carried out by one-way ANOVA and the post hoc Fisher's protected least significant differences test on the StatView program (SAS Institute, Cary, NC). Differences with P < 0.05 were considered significant.
 |
RESULTS
|
---|
Feeding stimulates pancreatic protein synthesis.
To validate the flooding-dose technique for measuring pancreatic protein synthesis in mice, we measured the uptake of radioactive phenylalanine into plasma and pancreas as a function of time. After the administration of phenylalanine, plasma phenylalanine increased
3.5-fold (to 252 ± 10 µM). The time course for the specific activity (dpm/nmol of L-Phe) of the pancreatic intracellular nonprotein pool is represented in Fig. 1A, and indicates that the precursor labeling in the pancreas pool does not change within the time range from 5 to 20 min after the injection of the tracer. The incorporation of L-[3H]Phe into pancreatic protein as a function of time is represented in Fig. 1B and shows a linear increase from 5 to 20 min. 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.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 1. Time course of specific radioactivity (A) and L-[3H]phenylalanine (Phe) incorporation into pancreatic protein (prot; B) in ICR mice. Values are expressed as disintegrations per minute per nanomole of Phe in the pancreatic acid soluble pool in A and as nanomoles of incorporated Phe per milligram of protein in the acid precipitable pool in B. All data points are means ± SE of 1012 mice per group.
|
|
Pancreatic protein synthesis determined at 9:0010:00 AM in mice fed ad libitum was 7.9 ± 0.3 nmol of L-Phe·10 min1·mg protein1. Fasting for 18 h reduced pancreatic protein synthesis to 70.8 ± 1.4% of control-fed animals (Fig. 2). Refeeding for 1 h increased protein synthesis to levels similar to the fed group, and refeeding for 2 h increased pancreatic protein synthesis to 117.3 ± 4.9% of the fed group or >50% compared with the fasted group (Fig. 2). Protein synthesis measured after 3 h refeeding returned to close to fasted levels; 77.4 ± 9.7% of control (Fig. 2). When the animals were fasted again for 6 h after a 2-h refeeding, protein synthesis levels had also returned to the fasted levels, being 68.2 ± 7.7% of control (Fig. 2). These results indicate that stimulating gastrointestinal activity in mice by feeding transiently stimulates pancreatic protein synthesis. Because increased stimulation of protein synthesis was seen after 1 and 2 h refeeding and was maximal after 2 h, we measured the effects of feeding on mRNA levels and the translational machinery primarily at the 2-h time point.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 2. Effect of fasting and feeding on Phe incorporation into pancreatic protein. Experimental groups are as described in MATERIAL AND METHODS with fasting for 18 h and refeeding at 9 AM. Values are expressed as nanomoles of incorporated phenylalanine per milligram of protein per 10 min and are means ± SE of 1220 mice/group. *P < 0.05 vs. fed group; #P < 0.05 vs. fasted.
|
|
Pancreatic mRNA levels of digestive enzymes were not changed by the feeding status.
To determine whether changes in protein synthesis might be due to transcriptional regulation, we evaluated the mRNA levels of nine digestive enzymes, three housekeeping proteins, and ribosomal 18 sRNA by quantitative real-time PCR. Total RNA was isolated and after purity was checked, equal amounts were used for RT, and a standard amount of cDNA was used as a template for PCR. Except for an effect of fasting to decrease and refeeding to increase back to the control for the housekeeping gene cyclophilin A, there were no significant changes between the housekeeping genes analyzed at any of the experimental conditions (Table 2). Only small differences were seen in the different treatments among amylase, chymotrypsin, elastase 1 and 2, triglyceride lipase, carboxyl ester lipase, and the three analyzed trypsin isoforms, with three increasing and five decreasing in response to refeeding. None of these changes was statistically significant (Table 2). Thus changes at the mRNA level cannot explain changes in protein synthesis seen during fasting and feeding (Fig. 2).
View this table:
[in this window]
[in a new window]
|
Table 2. Relative mRNA quantities of digestive enzymes and housekeeping genes in pancreas of animals fed ad libitum, fasted for 18 h, and refed for 2 h
|
|
The translation initiation factor eIF2B activity is not modified by the feeding status.
Because changes seen in pancreatic protein synthesis were not related to changes in mRNA for digestive enzymes, we analyzed the possible translation regulatory mechanisms that could be involved in this effect. Modulation of eIF2B activity is known to be an important regulatory point in translation initiation in some systems, because eIF2 is required for binding of initiator tRNA to the ribosome (16). Extracts of control-fed pancreas exhibited an eIF2B activity of 34.5 ± 3.3 nmol·min1·mg protein1 (n = 9). No significant reduction or increase of this activity was seen at any of the different experimental conditions (Fig. 3). We conclude that this regulatory step in translation initiation is active regardless of the feeding status, and thus it is not a limiting factor for normal pancreatic protein synthesis regulation.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 3. Effect of fasting and feeding on eIF2B activity. The results are expressed as a percentage of fed group levels. Experimental groups are similar to Fig. 2. Data shown are the means ± SE of 810 animals/group.
|
|
Feeding stimulates the Akt/mTOR pathway.
Because the Akt/mTOR pathway is known to be activated in the pancreas by physiological doses of CCK (47), we determined whether this stimulatory pathway was also activated by normal feeding in mice. Pancreatic Akt activity decreased after 18 h of fasting to 72.9 ± 9.4% of the fed group levels (Fig. 4A) and increased 1 (data not shown) and 2 h after refeeding to about 2.5 times the levels of the fasted group (Fig. 4A). These changes on Akt activity were correlated with changes in the phosphorylation status of the kinase on Ser473 (Fig. 4B). The feeding status did not change the total amount of the kinase (Fig. 4B, inset).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 4. Effect of fasting and feeding on Akt activity (A) and Akt phosphorylation on Ser473 (B). The results are expressed as a percentage of fed group levels. Data are the means ± SE of 1215 animals/group. In A, a representative blot for GSK3 substrate phosphorylation levels is shown in the inset. In B, representative blots for Akt phosphorylation on Ser473 and for total Akt are shown in the insets. *P < 0.05 vs. fed group, #P < 0.05 vs. fasted group.
|
|
To test whether mTOR was also activated, we measured the phosphorylation levels of two downstream target molecules, the ribosomal protein S6 and the 4E-BP1. The phosphorylation of the ribosomal protein S6 on Ser240/244 was used as a readout of S6 kinase activity; there was no change in the total amount of S6K in the experimental groups (data not shown). S6 phosphorylation was significantly decreased to 11.8 ± 3.2% of the fed group after 18 h of fasting and highly increased after refeeding to 394 ± 41.6% of fed levels (Fig. 5A). The phosphorylation of the 4E-BP1 was also significantly decreased on fasted animals and enhanced after refeeding for 2 h. Fasting reduced the highest phosphorylated
-form to 2.8 ± 0.7% of total compared with 14.6 ± 3.6% in the fed group (Fig. 5B). Refeeding for 2 h increased 4E-BP1 phosphorylation with 21.4 ± 3.0% of total in the
-form (Fig. 5B). Both, S6 and 4E-BP1 phosphorylation partially reversed following a 6-h fast after refeeding (data not shown). Phosphorylation of 4E-BP1 leads to the release of eIF4E, which participates in the formation of the eIF4F complex, another one of the main regulatory points on translation initiation. The association of the mRNA cap binding protein eIF4E with eIF4G by coimmunoprecipitation was measured as an indicator of eIF4F formation. The presence of the eIF4F complex was not significantly changed during fasting (Fig. 6) but was increased to 155 ± 13% (n = 4) of the fed control group after 1 h and to 170 ± 17% after 2 h of refeeding. All of these results together demonstrate that the Akt/mTOR pathway is activated by feeding. Both, the increased formation of eIF4F and the phosphorylation of ribosomal protein S6 stimulate the synthesis of digestive enzymes as well as that of ribosomal proteins and translation factors necessary for maintaining this acutely activated synthesis of digestive enzymes.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 6. Effects of fasting and feeding on the eIF4F complex formation, measured as eIF4E and eIF4G coimmunoprecipitation. Data for coimmunoprecipitated eIF4E and eIF4G are expressed as percentage of fed group levels and are the means ± SE of 812 animals/group. Representative blots for eIF4G associated with eIF4E and total eIF4E are shown in the insets. *P < 0.05 vs. fed group, # P < 0.05 vs. fasted group.
|
|
 |
DISCUSSION
|
---|
The present study was designed to evaluate the cellular mechanisms involved in the normal (meal to meal) stimulation of pancreatic digestive enzyme synthesis in mice. Fasting inhibited total pancreatic protein synthesis, and refeeding transiently increased it without changes in the mRNA levels of the digestive enzymes in any of these conditions. However, the increase in protein synthesis was accompanied by the activation of important regulatory points in protein translation (Fig. 7) that have been reviewed in detail elsewhere (38). Although the activity of the first regulatory point in translation initiation (eIF2B) was not modified by the feeding status, the formation of the eIF4F complex and the phosphorylation of 4E-BP1 and the ribosomal protein S6, downstream of the Akt/mTOR pathway, were enhanced after refeeding.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 7. G protein-coupled receptors stimulate translation initiation in pancreatic acinar cells through the phosphatidylinositol 3-kinase (PI3K) pathway. Scheme of the mechanisms that regulate translation initiation in pancreatic acinar cells. G protein-coupled receptors including CCK stimulate protein synthesis through the PI3K pathway. PI3K is activated in response to the active receptor and is believed to stimulate mTOR through phosphorylation of Akt/PKB, which, in turn, phosphorylates mTOR. The round red knobs denote regulatory phosphate groups. mTOR is responsible (at least in part) for phosphorylating the eIF4E binding protein 4E-BP1 that allows the release of the mRNA cap-binding protein eIF4E, which is required for the formation of the eIF4F complex, which also includes eIF4G (a scaffolding protein) and eIF4A (a RNA helicase) and is necessary for the global increase in translation. mTOR also phosphorylates directly or through another kinase (multiple arrows) S6K1 (p70S6k), which is responsible for phosphorylating ribosomal protein S6 (S6) and thereby increasing the translation of mRNAs with polypyrimidine tracts. These mechanisms were shown to be activated by meal feeding. eIF2 is required for Met-tRNA binding to the small ribosomal subunit. Phosphorylation of eIF2B (a GDP/GTP exchange factor) by GSK-3 results in inhibition of its GDP/GTP exchange activity. GSK-3 is itself inactivated (grey arrow) when it is phosphorylated by Akt/PKB. Thus the inactivation (phosphorylation) of GSK-3 causes dephosphorylation of eIF2B, leading to its activation, which enhances translation initiation. The phosphorylation of the -subunit of eIF2 on Ser51 by several stress-activated kinases can inhibit eIF2B activity. In the present study, meal feeding did not significantly alter eIF2B activity. In the figure, protein kinases are shown in blue, translation initiation factors in yellow, and scaffolding or structural proteins in purple.
|
|
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. The flooding-dose technique used in this study is based on the "flooding" of all precursor compartments with unlabeled amino acid (9, 12). Because accurate assessment of the specific radioactivity of the precursor amino acid at the site of protein synthesis is important, the administration of the labeled amino acid together with a large bolus of unlabeled amino acid makes the specific activities in all precursor pool compartments more alike than if the labeled compound is given as a tracer dose. The estimation of the precursor labeling specific activity from a readily accessible compartment pools such as the intracellular or plasma pool should, therefore, reflect that of the aminoacyl-tRNA, the direct precursor of protein synthesis, and remains almost constant for a certain period of time after injection (10).
We used L-[3H]Phe as a radioactive tracer because of its widespread use and because Sweiry and coworkers (45) demonstrated that phenylalanine transport into cells of the perfused rat pancreas was not a rate-limiting factor for protein synthesis. The flooding-dose technique has been well characterized in muscle (9, 11, 12), liver (7, 24), and heart (12), and in the present study, it has been adapted to measure pancreatic protein synthesis in mice. First, the specific activity of the intracellular unincorporated pancreatic compartment was measured at different time points to confirm that it remained almost constant for a certain period of time after injection (Fig. 1A) (12, 24, 45). Second, the actual incorporation of L-[3H]Phe into pancreatic protein at different time points was calculated (Fig. 1B). It was found that this parameter increased nearly linearly from 5 to 20 min after L-[3H]Phe administration, and an intermediate time point (10 min), well placed in the linear range and used elsewhere (12), was chosen to be used in all consecutive studies.
To match digestive enzyme secretion and synthesis to dietary need, synthesis of digestive enzymes needs to be controlled and the short-term meal-to-meal stimulation has to provide an immediate, reversible, and flexible response. Because
90% of pancreatic protein synthesis has been estimated to be devoted to digestive enzymes (34, 39), all of the results presented in this study implicitly reflect the changes in digestive enzyme synthesis after fasting and refeeding, and consequently, our discussion flows around this premise.
The results for total protein synthesis obtained for a 10-min period after 2 h of refeeding were higher than the rate after 1 and 3 h of refeeding (Fig. 2). One possible explanation for the maximal stimulation 2 h after refeeding is the delay between presentation of food and nutrients entering the small intestine. Another explanation could be that there is a delay between the stimulation of pancreatic secretion and the synthesis of new digestive enzymes after food intake (41); a decrease in hormonal and cholinergic stimulation after peak eating could correlate with the decreased protein synthesis found after 3 h. Fasting mice again for 6 h, after 2 h of refeeding, brought total protein synthesis levels down to the levels of the 18-h fasted animals, indicating that the pancreas can reach its "basal" synthetic point at this time after being stimulated by a meal.
Fasting is known to reduce protein synthesis in skeletal muscle and liver (1, 7), and refeeding stimulates it in the same organs (11, 18, 43, 44, 51). In the present study, we describe a reduction of total protein synthesis after 18 h of fasting in mouse pancreas, similar to what was found in rat liver and muscle in comparable experimental conditions (1, 7). These results are in agreement with a general effect of the feeding status on total protein synthesis of a variety of different organs whose activity can be more or less dependent on the animal's feeding status. The comparison of our results with other early pancreas studies (8, 46) is difficult, because those studies were carried out under very different experimental conditions (long fasting times, up to several days), different species of animals, and different methods to measure protein synthesis (i.e., in vitro measurements of total protein synthesis after in vivo treatment). However, the majority of these studies also showed reduced pancreatic protein synthesis with fasting (8).
The partial reduction of total protein synthesis seen in the present study with mice indicates that in the basal or interdigestive state, pancreatic protein synthesis (and consequently, digestive enzyme synthesis) is reduced but not completely blocked; it remains active to maintain certain levels of stored digestive enzymes and replace the ones that are still being secreted (41). Not surprisingly, the fractional rates of pancreatic protein synthesis are among the highest in mammalian tissues (45). Moreover, the lack of effect of fasting on Akt activity (Fig. 4), eIF2B activity (Fig. 2), and eIF4F complex formation (Fig. 6) compared with the control-fed group would be in concordance with keeping the synthetic machinery active under these conditions. Because it is known that CCK and cholinergic analogs stimulate these factors at submaximal doses (38), it could likely be that interdigestive (basal) stimulation of the pancreas by the cholinergic nervous system and continuous (or phasic) secretion of CCK (41) maintained the activity of these factors that could account for the synthesis of digestive enzymes during fasting.
In the same conditions (18 h fasting), mRNA levels of the studied housekeeping genes and digestive enzymes did not change, compared with the control-fed group. These results clearly indicate that the regulation of digestive enzyme synthesis in fasting has to be mainly related to changes in the translation of mRNAs into protein. Along these lines, the increase of total protein synthesis seen after refeeding for 1 and 2 h, compared with the fasted group, without an increase of the digestive enzymes mRNA levels, indicates that the stimulation of pancreatic protein synthesis after food intake is also regulated at the translational level.
Although the results from early studies were conflicting (8), those from Black and Webster (3) and Morisset and Webster (29) indicated that translational control was involved in the regulation of pancreatic protein synthesis by feeding. Only now, however, a clear involvement of specific translation factors has been described. In mouse pancreas, protein synthesis levels were enhanced after 2 h of refeeding and correlated with activation of the Akt/mTOR pathway, as shown by the increase in Akt activity and phosphorylation, the phosphorylation levels of two downstream effectors of mTOR, 4E-BP1 and the ribosomal proteins S6, and in the formation of the eIF4F complex. The activation of the Akt/mTOR pathway will lead to an increased phosphorylation of S6 and 4E-BP1 and formation of the eIF4F complex that binds the capped mRNA to start translation (13) similar to what has been described in muscle and liver (18). The large phosphorylation of the S6 ribosomal protein in this study indicates that the synthesis of more translational machinery and ribosomal proteins (27) is activated by refeeding. It is known that the phosphorylation of S6 can be regulated by CCK (4, 5, 36), insulin (42), and the branched-chain amino acid leucine (37) in rat pancreas as has been shown in other organs (2, 23, 49). Cholinergic stimulation and insulin have been demonstrated to activate protein synthesis in pancreas (21, 28, 48) and the translational machinery in other organs (15, 32, 48) as well. Thus the stimuli involved in the food intake process that increases vagal neural activity, plasma CCK levels and protein and aminoacids from the food itself (41), could each contribute to the stimulation of pancreatic protein synthesis (14, 22) and the activation of translational machinery in the exocrine pancreas after a meal. The lack of effect on pancreatic eIF2B activity by the feeding status has also been described in liver and muscle (11, 50). In our case, this "noneffect" may be explained by the already high basal activity of eIF2B in pancreas (35) that was not altered by fasting and/or feeding.
In conclusion, normal feeding in ICR mice stimulates pancreatic protein synthesis by stimulating the translational machinery through the Akt/mTOR pathway, without changes at the mRNA level. Because food intake triggers several gastrointestinal stimuli that are known to both stimulate pancreatic exocrine secretion and affect the translational machinery, it is very likely that all of them, at least to some extent, play a role in the stimulation of pancreatic protein synthesis in this situation. More studies are necessary to elucidate the contribution of each of these factors in the integrated regulation of pancreatic protein synthesis that occurs in response to feeding.
 |
GRANTS
|
---|
This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52860, DK-59578 (to J. A. Williams), and DK-34933 (to the Michigan Gastrointestinal Peptide Center) and the Michigan Center for Integrative Genomics.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Scott Kimball of Pennsylvania State Medical School for generous provision of reagents and ongoing advice on translational control; Richard Mortensen and Debora Vanheyningen of University of Michigan, for helpful advice with the Q-RT-PCR studies; and Qun Xie and Steve 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 and jawillms{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.
 |
REFERENCES
|
---|
- Anthony JC, Anthony TG, Kimball SR, Vary TC, and Jefferson LS. Orally administered leucine stimulates protein synthesis in skeletal muscle of postabsorptive rats in association with increased eIF4F formation. J Nutr 130: 139145, 2000.[Abstract/Free Full Text]
- Anthony JC, Yoshizawa F, Anthony TG, Vary TC, Jefferson LS, and Kimball SR. Leucine stimulates translation initiation in skeletal muscle of postabsorptive rats via a rapamycin-sensitive pathway. J Nutr 130: 24132419, 2000.[Abstract/Free Full Text]
- Black O Jr. and Webster PD. Protein synthesis in pancreas of fasted pigeons. J Cell Biol 57: 18, 1973.[Abstract/Free Full Text]
- Bragado M, Groblewski G, and Williams J. Regulation of protein synthesis by cholecystokinin in rat pancreatic acini involves PHAS-I and the p70 S6 kinase pathway. Gastroenterology 115: 733742, 1998.[ISI][Medline]
- Bragado MJ, Groblewski GE, and Williams JA. p70s6k is activated by CCK in rat pancreatic acini. Am J Physiol Cell Physiol 273: C101C109, 1997.[Abstract/Free Full Text]
- Bragado MJ, Tashiro M, and Williams JA. Regulation of the initiation of pancreatic digestive enzyme protein synthesis by cholecystokinin in rat pancreas in vivo. Gastroenterology 119: 17311739, 2000.[ISI][Medline]
- Burrin DG, Davis TA, Fiorotto ML, and Reeds PJ. Hepatic protein synthesis in suckling rats: effects of stage of development and fasting. Pediatr Res 31: 247252, 1992.[Abstract]
- Case RM. Synthesis, intracellular transport and discharge of exportable proteins in the pancreatic acinar cell and other cells. Biol Rev Camb Philos Soc 53: 211354, 1978.[ISI][Medline]
- Caso G, Ford GC, Nair KS, Garlick PJ, and McNurlan MA. Aminoacyl-tRNA enrichment after a flood of labeled phenylalanine: insulin effect on muscle protein synthesis. Am J Physiol Endocrinol Metab 282: E1029E1038, 2002.[Abstract/Free Full Text]
- Davis TA, Fiorotto ML, Nguyen HV, and Burrin DG. Aminoacyl-tRNA and tissue free amino acid pools are equilibrated after a flooding dose of phenylalanine. Am J Physiol Endocrinol Metab 277: E103E109, 1999.[Abstract/Free Full Text]
- Davis TA, Nguyen HV, Suryawan A, Bush JA, Jefferson LS, and Kimball SR. Developmental changes in the feeding-induced stimulation of translation initiation in muscle of neonatal pigs. Am J Physiol Endocrinol Metab 279: E1226E1234, 2000.[Abstract/Free Full Text]
- Garlick PJ, McNurlan MA, and Preedy VR. A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [3H]phenylalanine. Biochem J 192: 719723, 1980.[ISI][Medline]
- Gingras A, Raught B, and Sonenberg N. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 68: 913963, 1999.[CrossRef][ISI][Medline]
- Graf R, Valeri F, Gassmann R, Hailemariam S, Frick TW, and Bimmler D. Adaptive response of the rat pancreas to dietary substrates: parallel regulation of trypsinogen and pancreatic secretory trypsin inhibitor. Pancreas 21: 181190, 2000.[CrossRef][ISI][Medline]
- Guizzetti M and Costa LG. Activation of phosphatidylinositol 3 kinase by muscarinic receptors in astrocytoma cells. Neuroreport 12: 16391642, 2001.[CrossRef][ISI][Medline]
- Kimball SR. Eukaryotic initiation factor eIF2. Int J Biochem Cell Biol 31: 2529, 1999.[CrossRef][ISI][Medline]
- Kimball SR, Everson WV, Flaim KE, and Jefferson LS. Initiation of protein synthesis in a cell-free system prepared from rat hepatocytes. Am J Physiol Cell Physiol 256: C28C34, 1989.[Abstract/Free Full Text]
- Kimball SR, Jefferson LS, Nguyen HV, Suryawan A, Bush JA, and Davis TA. Feeding stimulates protein synthesis in muscle and liver of neonatal pigs through an mTOR-dependent process. Am J Physiol Endocrinol Metab 279: E1080E1087, 2000.[Abstract/Free Full Text]
- Korc M. Regulation of pancreatic protein synthesis by cholecystokinin and calcium. Am J Physiol Gastrointest Liver Physiol 243: G69G75, 1982.[Abstract/Free Full Text]
- Korc M, Bailey A, and Williams JA. Regulation of protein synthesis in normal and diabetic rat pancreas by cholecystokinin. Am J Physiol Gastrointest Liver Physiol 241: G116G121, 1981.[Abstract/Free Full Text]
- Korc M, Iwamoto Y, Sankaran H, Williams JA, and Goldfine ID. Insulin Action in Pancreatic Acini from Streptozotocin-Treated Rats. I. Stimulation of protein synthesis. Am J Physiol Gastrointest Liver Physiol 240: G56G62, 1981.[Abstract/Free Full Text]
- Leroy J, Morisset JA, and Webster PD. Dose-related response of pancreatic synthesis and secretion to cholecystokinin-pancreazymin. J Lab Clin Med 78: 149157, 1971.[ISI][Medline]
- Liu Z, Jahn LA, Wei L, Long W, and Barrett EJ. Amino acids stimulate translation initiation and protein synthesis through an Akt-independent pathway in human skeletal muscle. J Clin Endocrinol Metab 87: 55535558, 2002.[Abstract/Free Full Text]
- Lundholm K, Ternell M, Zachrisson H, Moldawer L, and Lindstrom L. Measurement of hepatic protein synthesis in unrestrained mice-evaluation of the flooding technique. Acta Physiol Scand 141: 207219, 1991.[ISI][Medline]
- Mathews M, Sonenberg N, and Hershey JW. Origins and principles of translational control. In: Translational Control of Gene Expression, edited by Sonenberg N, Hershey JW, and Mathews M. New York: Cold Spring Harbor Laboratory Press, 2000, p. 131.
- Meyuhas O. Synthesis of the translational apparatus is regulated at the translational level. Eur J Biochem 267: 63216330, 2000.[Abstract/Free Full Text]
- Meyuhas O and Hornstein E. Translational control of TOP mRNAs. In: Translational Control of Gene Expression, edited by Sonenberg N, Hershey JW, and Mathews M. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2000, p. 671693.
- Morisset JA, Black O Jr, and Webster PD. Effects of fasting, feeding, and bethanechol chloride on pancreatic microsomal protein synthesis in vitro 1. Proc Soc Exp Biol Med 140: 13081314, 1972.
- Morisset JA and Webster PD. Effects of fasting and feeding on protein synthesis by the rat pancreas. J Clin Invest 51: 18, 1972.[ISI][Medline]
- Mothe-Satney I, Yang D, Fadden P, Haystead TA, and Lawrence JC. Multiple mechanisms control phosphorylation of PHAS-I in five (S/T) sites that govern translational repression. Mol Cell Biol 20: 35583567, 2000.[Abstract/Free Full Text]
- Okabayashi Y, Moessner J, Logsdon CD, Goldfine ID, and Williams JA. Insulin and other stimulants have nonparallel translational effects on protein synthesis. Diabetes 36: 10541060, 1987.[Abstract]
- Proud CG and Denton RM. Molecular mechanisms for the control of translation by insulin. Biochem J 328: 329341, 1997.[ISI][Medline]
- Rausch U, Rudiger K, Vasiloudes P, Kern H, and Scheele G. Lipase synthesis in the rat pancreas is regulated by secretin. Pancreas 1: 522528, 1986.[Medline]
- Rinderknecht H. Pancreatic secretory enzymes. In: The Pancreas. Biology, pathobiology and disease (2nd ed.), edited by Go VLW, DiMagno EP, Gardner JD, Lebenthal E, Reber HA, and Scheele GA. New York: Raven, 1993, p. 219251.
- Sans M, Kimball S, and Williams J. Effect of CCK and intracellular calcium to regulate eIF2B and protein synthesis in rat pancreatic acinar cells. Am J Physiol Gastrointest Liver Physiol 282: G267G276, 2002.[Abstract/Free Full Text]
- Sans MD, DiMagno MJ, D'Alecy LG, and Williams JA. Caerulein-induced acute pancreatitis inhibits protein synthesis through effects on eIF2B and eIF4F. Am J Physiol Gastrointest Liver Physiol 285: G517G528, 2003.[Abstract/Free Full Text]
- Sans MD, Tashiro M, Samuelson LC, D'Alecy LG, Kimball SR, and Williams JA. L-Leucine activates pancreatic translational effectors in rat and mouse pancreas (Abstract). Gastroenterology 120: A-335, 2001.
- Sans MD and Williams JA. Translational control of protein synthesis in pancreatic acinar cells. Int J Gastrointest Cancer 31: 107115, 2002.[CrossRef][ISI][Medline]
- Scheele GA. Regulation of pancreatic gene expression in response to hormones and nutritional substrates. In: The Pancreas. Biology, Pathobiology and Disease (2nd ed.), edited by Go VLW, DiMagno EP, Gardner JD, Lebenthal E, Reber HA, and Scheele GA. New York: Raven, 1993, p. 103120.
- Schick J, Kern H, and Scheele G. Hormonal stimulation in the exocrine pancreas results in coordinate and anticoordinate regulation of protein synthesis. J Cell Biol 99: 15691574, 1984.[Abstract]
- Singer MV. Neurohormonal control of pancreatic enzyme secretion in animals. In: The Pancreas. Biology, Pathobiology and Disease (2nd ed.), edited by Go VLW, DiMagno EP, Gardner JD, Lebenthal E, Reber HA, and Scheele GA. New York: Raven, 1993, p. 425448.
- Sung CK and Williams JA. Insulin and ribosomal protein S6 kinase in rat pancreatic acini. Diabetes 38: 544549, 1989.[Abstract]
- Svanberg E, Ennion S, Isgaard J, and Goldspink G. Postprandial resynthesis of myofibrillar proteins is translationally rather than transcriptionally regulated in human skeletal muscle. Nutrition 16: 4246, 2000.[CrossRef][ISI][Medline]
- Svanberg E, Ohlsson C, Hyltander A, and Lundholm KG. The role of diet components, gastrointestinal factors, and muscle innervation on activation of protein synthesis in skeletal muscles following oral refeeding. Nutrition 15: 257266, 1999.[CrossRef][ISI][Medline]
- Sweiry JH, Emery PW, Munoz M, Doolabh K, and Mann GE. Influx and incorporation into protein of L-phenylalanine in the perfused rat pancreas: effects of amino acid deprivation and carbachol. Biochim Biophys Acta 1070: 135142, 1991.[ISI][Medline]
- Webster PD 3rd, Black O Jr, Mainz DL, and Singh M. Pancreatic acinar cell metabolism and function. Gastroenterology 73: 14341449, 1977.[ISI][Medline]
- Williams JA. Intracellular signaling mechanisms activated by cholecystokinin-regulating synthesis and secretion of digestive enzymes in pancreatic acinar cells. Annu Rev Physiol 63: 7797, 2001.[CrossRef][ISI][Medline]
- Williams JA and Goldfine ID. The insulin-pancreatic acinar axis. Diabetes 34: 980986, 1985.[Abstract]
- Xu G, Kwon G, Cruz WS, Marshall CA, and McDaniel ML. Metabolic regulation by leucine of translation initiation through the mTOR-signaling pathway by pancreatic beta-cells. Diabetes 50: 353360, 2001.[Abstract/Free Full Text]
- Yoshizawa F, Kimball SR, and Jefferson LS. Modulation of translation initiation in rat skeletal muscle and liver in response to food intake. Biochem Biophys Res Commun 240: 825831, 1997.[CrossRef][ISI][Medline]
- Yoshizawa F, Kimball SR, Vary TC, and Jefferson LS. Effect of dietary protein on translation initiation in rat skeletal muscle and liver. Am J Physiol Endocrinol Metab 275: E814E820, 1998.[Abstract/Free Full Text]
Copyright © 2004 by the American Physiological Society.