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* Department of Molecular and Integrative Physiology and Department of Internal Medicine, The University of Michigan Medical School, Ann Arbor, MI 48109-0622 and ** Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA 17033
4 To whom correspondence should be addressed. E-mail: mdsansg{at}umich.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • pancreas • leucine • translation • cholecystokinin • insulin
Nutritional signals play an important role in controlling gene expression in mammals, with both major and minor dietary constituents participating in the regulation of gene expression (1,2). In recent years, it has become clear that mRNA translation, in addition to transcription, represents an important control point in gene expression (3,4). Nutrients have a profound effect on translation and amino acids. Specifically, they have important regulatory roles in the control of mRNA translation in skeletal muscle and liver in addition to their role as substrates for protein synthesis (5,6). Of all amino acids, the BCAAs leucine, isoleucine, and valine have a unique role in this process (7) as shown using in vitro and in vivo models (5,8–11). Among BCAA, leucine appears to have the highest ability to augment key events in translation initiation and in some tissues to stimulate protein synthesis (8,12,13).
Amino acids activate at least 2 signaling pathways, a rapamycin-sensitive pathway involving the mammalian target of rapamycin (mTOR,6 a serine/threonine kinase) and an unknown rapamycin-insensitive pathway (13,14). mTOR serves as a convergence point for signaling by growth factors and amino acids to the mRNA binding step of translation initiation (15,16) and regulates the phosphorylation of the translational repressor factor 4E (eIF4E) binding protein 1 (4E-BP1) and of the 70-kDa ribosomal protein S6 kinase (S6K) (17). mTOR is also believed to be upstream of the upregulation of the translation of the 5'-terminal oligopyrimidine tract mRNA species, which encode ribosomal proteins and elongation factors (18).
In skeletal muscle, oral administration of BCAA increases the availability of eukaryotic initiation factor 4E (eIF4E), a protein that binds to the cap structure present at the 5'-end of the mRNA, as part of the eIF4F complex which includes eIF4G, a large polypeptide that functions as a scaffold (12). The increase in eIF4E availability is due in part to the leucine-dependent hyperphosphorylation of its translational repressor, 4E-BP1, which decreases its affinity for eIF4E, thereby facilitating the association of eIF4E with eIF4G (19). Increased activity of S6K was also implicated in stimulating protein synthesis under conditions that also promote 4E-BP1 phosphorylation (12).
With each meal, the pancreas must synthesize new digestive enzymes to replace those secreted, and this occurs without significant change in the digestive enzyme mRNA content (20). In isolated pancreatic acini, secretagogues including cholecystokinin (CCK) and cholinergic analogs, as well as insulin, stimulate the synthesis of digestive enzymes at the translational level (21–24). The phosphoinositide 3-kinase (PI3K)/mTOR pathway was shown to be of primary importance for protein synthesis at the mRNA translation level in rat pancreatic acini stimulated by CCK in vitro in that CCK stimulates the phosphorylation of 4E-BP1 and S6K through a PI3K and mTOR dependent pathway (25,26).
The present study was designed to elucidate whether oral gavage of BCAA would stimulate the protein translation machinery and protein synthesis in the exocrine pancreas.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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/ß (Ser-21/9), ribosomal protein S6, phospho-S6 (Ser-240/244), S6K, and phospho-S6K (Thr-389) were from Cell Signaling; eIF4E-BP polyclonal antibody was from Calbiochem. Rabbit anti-eIF4G antibody was a gift from Dr. R. E. Rhoads (LSU). Goat anti-rabbit and anti-mouse Ig G conjugated to horseradish peroxidase and ECL reagent were from Amersham Pharmacia Biotech. Animals and experimental design. All animal studies were approved by the University of Michigan Committee on Use and Care of Animals. Male Sprague-Dawley rats (Harlan Sprague Dawley), with a mean weight of 170.3 ± 4.6 g, were food deprived for 18 h before experimentation. In the first study, rats were randomly divided into 5 groups and administered L-leucine, L-valine, L-isoleucine, L-phenylalanine, or vehicle by i.g. gavage with a 16-gauge stainless steel feeding tube without anesthesia at 1.35 mg/g body weight (BW) and a volume of 1.5 mL/100 g BW (8,12) in an aqueous solution (pH 2.5). The amount of amino acids given was equivalent to the amount of leucine consumed by rats during 24 h of free access to an AIN-93 powdered diet (27). Phenylalanine (as well as L-threonine) was administered to demonstrate the specificity of the effects of the BCAA. Control rats were administered acidified aqueous solution at the same volume ratio. Thirty minutes after gavage, blood was collected by cardiac puncture after CO2 asphyxiation, and pancreas samples were removed and homogenized in specified lysis buffer (28). In a second study, after leucine gavage at 1.35 mg/g BW, rats were returned to their cages with free access to water and after 15, 30, 60, and 90 min, blood and pancreas was collected. For the third study, the mTOR inhibitor rapamycin was administered by i.v. injection at a dose of 0.75 mg/kg and 1 mL/100 g BW, 2 h before the oral administration of leucine (1.35 mg/g BW). Vehicle (0.9% sodium chloride with 2% ethanol) was administered to the rapamycin control groups.
For studies in mice, male ICR mice (Harlan Sprague Dawley), weighing 
30 g, and female CCK-deficient (CCK/KO) or genetically matched control 129/Sv mice, weighing 20 g, were used. The mice were gavaged using a 18-gauge stainless steel feeding tube under brief isoflurane anesthesia and given either L-leucine suspension (1.35 mg/g BW) in 0.5% carboxymethylcellulose (wt:v) in distilled water, as vehicle or vehicle alone (0.3 mL). One hour after the treatment, mice were killed by CO2 asphyxiation and decapitation, and trunk blood and pancreas were collected.
In a separate set of experiments, diabetes was induced in male Sprague-Dawley rats with an i.p. injection of streptozotocin (22). Diabetic rats were used after 4–6 d when their glucose plasma levels were higher than 450 mg/dL (25 mmol/L), to obtain an insulin-free system for experiments in vivo and for the preparation of isolated pancreatic acini on in vitro experiments.
Preparation of pancreatic acini and cell lysates. Pancreatic acini were prepared by collagenase digestion of pancreas from normal and diabetic male Sprague-Dawley rats weighing 125–150 g (24,29). Acini were suspended and preincubated in buffer, consisting of an N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid-buffered Ringer solution supplemented with 11.1 mmol/L glucose, Eagle's minimal essential amino acids mixture at a final concentration (µmol/L) of: L-arginine (726), L-cystine (198), L-histidine (271), L-isoleucine (399), L-leucine (399), L-lysine (420), L-methionine (101), L-phenylalanine (200), L-threonine (400), L-tryptophan (499), L-tyrosine (199), L-valine (399), and L-glutamine (2000); 0.1 g/L soybean trypsin inhibitor and 1 g/L bovine serum albumin and gassed with 100% O2. After 1 h preincubation at 37°C, acini were incubated for 30 min with or without leucine at 1100 µmol/L in fresh buffer at 37°C. Alternatively, acini were preincubated for 1 h in the same previously described buffer but without any of the 3 BCAA. After preincubation, acini were resuspended in fresh buffer with 100% O2, distributed in 2-mL aliquots and incubated at 37°C (24) with or without leucine at 1100 µmol/L, isoleucine at 1200 µmol/L, and valine at 2100 µmol/L for 30 min. These amino acid concentrations were equivalent to their respective concentration in rat plasma samples after 30 min of gavage in vivo (Table 1). Acinar samples were then collected for Western blotting (29).
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Assembly of the eIF4F complex. To analyze the assembly of the eIF4F complex, we determined the association of eIF4E with eIF4G by quantitating the amount of eIF4G bound to immunoprecipitated eIF4E using specific anti-eIF4E antibody, as previously described (25,30,31).
Measurement of eIF2B activity. 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).
Measurements of plasma amino acids by HPLC. Plasma samples were prepared as described previously (30). Fluorescent derivatives for amino acids were prepared by reacting samples to the fluorescent derivatizing agent, AccQ*Fluor (Waters) in pH 8.8 borate buffer. Valine, isoleucine, leucine, and phenylalanine in standards and unknowns were quantified by reversed-phase LC (Millennium32 v3.2 Chromatographic Workstation, Waters). A two-pump gradient system delivered 10 mmol/L sodium acetate trihydrate (pH 5.0; mobile phase A) and 60% acetonitrile in HPLC grade water (mobile phase B) at 1 mL/min for 48 min. The gradient (described as a percentage of the mobile phase B) was as follows: 10% at 0 min; 35% at 33 min; 100% 34–37 min; and 10% 38–48 min. Separation was performed on a 4.6 mm x 150 mm, 3.5 µm column (Waters, XterraMS C18) with an identical guard column, both at 36°C. Standards, blanks, and samples were injected and fluorescent peak height and area evaluated at an excitation of 250 nm and an emission of 395 nm using a fluorescence detector.
Measurement of pancreatic protein synthesis. Rat pancreatic protein synthesis was determined using the flooding dose technique as described previously by us for mice (20,30). Briefly, 14.8 µBq/g of L-[3H]Phe together with unlabeled L-Phe (1.5 µmol/g) was injected into the tail vein. In preliminary studies, we validated this method for rat experiments and found that the uptake of L-phenylalanine into the pancreas and its specific activity in the perchloric acid (PCA)-soluble pool was nearly constant from 5 to 20 min after the injection of the radioactive tracer. We chose 15 min as the optimal time to determine protein synthesis, because incorporation of 3H-Phe into protein was increasing linearly at this time point. Rats were killed and the pancreas rapidly removed and frozen in liquid nitrogen. Frozen pancreas was subsequently homogenized in 10 volumes of 0.6 mol/L PCA and processed as described previously (20). L-Phe was measured by HPLC and protein synthesis was expressed as nmol 3H-L-Phe/mg protein.
Measurement of plasma insulin. Plasma insulin concentration was determined at the Michigan Diabetes Research and Training Center using a modification of the insulin assay described by Linco Research. Insulin measurements were performed by double-antibody RIA using a 125I-human insulin tracer and a guinea pig anti-rat insulin first antibody from Linco Research; a rat insulin standard from Novo Research Institute; and a sheep anti-guinea pig gamma globulin secondary antibody, with 3% of PEG 6000 added, developed in the same center. The limit of sensitivity for the assay is 1 mU/L. The interassay and intra-assay variabilities were 11.5 and 3.2%, respectively, at 28.5 mU/L.
Statistical analysis. Data from rat experiments are represented as means and SEM and were obtained from 2–4 different experiments with 3–5 rats studied per group. Mouse results are represented as means and SEM, n = 4 mice/group. Results from in vitro studies were obtained from 4 different experiments (each of which used 1 rat) with each condition studied in duplicate in each experiment. Statistical analysis was carried out by one-way ANOVA; when a significant overall effect was detected, differences among individual means were assessed with Tukey's Multiple Comparison Test. In Figures 4 through 7, two-way ANOVA was applied; when a significant interaction or overall effect was detected, differences among individual means were assessed with Tukey's Multiple Comparison Test. If no significant interaction was detected, a 1-way ANOVA was performed. GraphPad PRISM3 software was used for statistical analysis. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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4-fold of control to 1093 µmol/L, isoleucine and valine increased 10-fold to 1226 and 2152 µmol/L, respectively, and phenylalanine concentration increased 7-fold to 540 µmol/L (Table 1). When a particular amino acid was given, the plasma levels of the other BCAA and phenylalanine tended to decrease.
All 3 BCAA increased the phosphorylation of the eIF4E binding protein (4E-BP1), although leucine had the strongest effect, increasing the percentage of the 
-form from 11.5 ± 2.2 to 52.2 ± 4.1 of the total after 30 min (Fig. 1A). The non-BCAA phenylalanine did not affect 4E-BP1 and S6K phosphorylation (Fig. 1A). The effect of leucine on the phosphorylation of the ribosomal protein S6 kinase (S6K) on Thr 389, indicative of its activation, was much greater, expressed in arbitrary units (21.4 ± 5.1 AU) than that of isoleucine (7.9 ± 2.2 AU) and valine (8.4 ± 3.3 AU) all compared with 0.04 ± 0.03 AU for the control group (Fig. 1B). Phenylalanine did not increase S6K phosphorylation (Fig. 1B). Threonine at the same dose also had no effect on 4E-BP1 or S6K phosphorylation (data not shown). These results clearly indicate that BCAA can stimulate phosphorylation of biomarkers of mRNA translation in rat pancreas; of the amino acids examined, leucine had the largest effect. Whether isoleucine and valine have additional effects on S6K in addition to activating the mTOR pathway remains to be determined.
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form, from 6.7 ± 1.0 to 46.5 ± 2.6% of total (Supplemental Fig. 1A) and the phosphorylation of S6K, from 0.9 ± 0.3 to 23.0 ± 6.3 AU (Supplemental Fig. 1B). These results indicate that oral administration of the same dose of leucine results in similar plasma levels in mice and rats, and has similar stimulatory effects on 4E-BP1 and S6K phosphorylation in the pancreas. To elucidate whether CCK could be involved in the stimulatory effects of leucine in protein translation, we gavaged leucine into control (wild-type) mice and mice lacking CCK (CCK/KO). The effects of leucine gavage on the formation of the eIF4F complex were not altered in CCK/KO mice compared with the control group (Fig. 4). Leucine also stimulated phosphorylation of 4E-BP1 in both control 129/Sv mice and in CCK/KO mice (data not shown). These results clearly indicate that leucine stimulation of the translational machinery in the exocrine pancreas is independent of CCK.
Effects of BCAAs are independent of insulin. To assess whether insulin could mediate some of the effects observed after gavaging with the BCAA (32), and because insulin was also involved in the stimulation of the translational machinery of the exocrine pancreas (24,33), we analyzed plasma insulin levels 30 min after amino acid gavage and compared them with postprandial levels. Valine and phenylalanine did not increase insulin levels over control values, but leucine and isoleucine increased plasma insulin to 2-fold that of control (Table 2). These values, however, were lower than the postprandial insulin plasma levels 30 min or 1 h after a meal that followed an 18-h period of food deprivation (Table 2).
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-form decreased, and the addition of each individual amino acid increased 4E-BP1 phosphorylation in acini (Fig. 5A). The effect of BCAA readdition to deprived acini on S6 phosphorylation showed a pattern similar to that for 4E-BP1 (Fig. 5B) although there was no effect when BCAA were added to the medium of acini incubated in HR buffer with a complete mixture of essential amino acids. Similar results for 4E-BP1 and S6 phosphorylation were obtained when experiments were performed in isolated acini from control, nondiabetic rats (data not shown). These results taken together indicate that despite the increase in plasma insulin after leucine and isoleucine administration to rats in vivo, the BCAA can stimulate the exocrine pancreas translational machinery independently of insulin. Leucine effects in rat pancreas are dependent on mTOR. The mTOR inhibitor rapamycin completely blocked the leucine-induced increase in phosphorylation of both proteins, 4E-BP1 (Fig. 6A) and the ribosomal protein S6 (Fig. 6B).
Akt activity (an upstream regulator of mTOR) was also analyzed and there was an increase of 468 ± 108% over control 30 min after leucine gavage in normal rats (n = 8; P < 0.05). To analyze whether this activation of Akt could be due to an increase in insulin levels, we first measured Akt phosphorylation on Ser 473 (indicative of its activation) in normal and diabetic rats gavaged with leucine for 30 min. Akt phosphorylation was increased in normal rats from 7.9 ± 3.4 to 14 ± 0.4 AU after leucine gavage and in diabetic rats from 6.2 ± 2 to 11.9 ± 2.6 AU (n = 4–8 rats per group; P < 0.05). Thus, Akt activation is independent of an increase in insulin levels in vivo. Second, we analyzed the same parameter in experiments with isolated acini from diabetic rats; all 3 BCAA enhanced Akt phosphorylation on Ser 473 when added back to acini that had the BCAA removed from the incubation media (Fig. 7). Similar results were seen in acini prepared from normal rats (data not shown). We conclude that the BCAA stimulate Akt independently of insulin, and that leucine effects on the translational machinery of rat pancreas are dependent on mTOR.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Nutritional status is important for pancreatic physiology, with pancreatic function known to be altered by a number of nutritional factors, including changes in major dietary components, protein, carbohydrates, and fat (36). We showed recently that feeding a balanced diet stimulates pancreatic protein synthesis in mice (20), and it was demonstrated that changes in diet composition affect the type of digestive enzymes that are synthesized and secreted (37). It was also shown that feeding a high-protein diet to mice (38) or a high–amino acid diet to rats (39) induced pancreatic hypertrophy and increased proteolytic enzyme content in the absence of CCK. These effects could likely be due to an increase in BCAA from the high-protein and high–amino acid diet that would stimulate the translational machinery leading to an increase in the pancreatic proteolytic enzyme content as well as nonzymogen proteins that would finally lead to hypertrophy.
BCAA, and leucine in particular, were reported to stimulate protein synthesis and the translational machinery in skeletal muscle (6,12,40). In the present study, all 3 BCAA stimulated the phosphorylation of eIF4E, its binding protein 4E-BP1, the ribosomal protein S6 kinase, and the formation of the eIF4F complex in rat pancreas but to different extents, as reported previously in other tissues. Although the stimulation of these translation factors usually indicates activation of total protein synthesis, this is not always the case (35). In our study, BCAA did not stimulate total protein synthesis, indicating that BCAA by themselves are not sufficient to stimulate global rates of protein synthesis in the pancreas. BCAA could be involved in the translation of a subset of proteins, characterized by the presence of an oligopyrimidine tract at the immediate 5' end (referred to as TOP) which are involved in the translation of ribosomal proteins and translation factors (18). In this scenario, amino acids would "prime" the pancreatic translational machinery for protein translation to occur, but a hormonal and/or cholinergic stimulus would be required for a complete stimulation of protein synthesis that might also include translation elongation steps. Moreover, feeding an individual amino acid might induce an imbalance in the pancreatic amino acid pool, which would restrict the availability of substrates for the synthesis of new proteins and thus create a limiting factor for zymogen synthesis. The latter idea is supported by the observation that administration of a single BCAA lowered the plasma concentration of other essential amino acids (Table 1) and by the fact that leucine stimulates protein synthesis in skeletal muscle (6,12,40); this might create a decrease in the availability of amino acids in the pancreatic pool.
In addition to the aforementioned translation factors, in some tissues, eIF2B activity is also believed to increase total protein synthesis (2,41). In pancreatic acinar cells, eIF2B activity is already high under resting conditions and it is not further increased by CCK at concentrations that stimulate protein synthesis in isolated acini (28). Our current results show no stimulatory effect of leucine on eIF2B activity in the pancreas and although it was shown that insulin stimulates eIF2B activity in different tissues (42,43), the increased plasma insulin levels due to leucine gavage did not modify its activity in pancreas. Moreover, although leucine stimulates eIF2B activity in leucine-deprived L6 myoblasts (44), neither amino acid perfusion of the gastrocnemius (45) nor oral leucine gavage in rat skeletal muscle (12) modified its activity. This regulatory step seems to be affected by severe amino acid limitation rather than by excess. From our results, eIF2B activity is likely not responsible for the leucine-induced stimulation of mRNA translation initiation.
We showed previously that feeding (20), CCK, cholinergic agonists, and insulin stimulated the translational machinery and total protein synthesis in murine exocrine pancreas in vivo (31) and in vitro (24,25,28,29,46). In the present study, we demonstrated that BCAA similarly activated key translation factors (4E-BP1, S6K, eIF4E, and eIF4F) in mouse pancreas in vivo, indicating that its effect is not species specific. Leucine effects were independent of CCK input, as demonstrated by leucine stimulation of regulatory translation factors in CCK-deficient mice. Leucine could "prime" the pancreatic synthetic machinery postprandially to synthesize more digestive enzymes without the need of a CCK stimulus, and/or could be activating selectively the synthesis of proteins important for intracellular metabolism (18).
Leucine stimulates insulin production in ß-cells (16,47); consequently, it increases plasma insulin levels that can mediate the enhancement of protein synthesis in skeletal muscle after ingestion of a balanced meal in postabsorptive rats (32). Insulin can activate translation in different tissues (19,42) including the pancreas (22,33), and the increase in 4E-BP1 and S6K phosphorylation reported in this study after leucine gavage could be due to this increase of plasma insulin levels. Rat plasma insulin levels measured after leucine gavage were similar to those reported by Anthony et al. (32), but were only 50% of the postprandial levels obtained 30 min after eating a full meal. To confirm that the increased levels of insulin were not additive or interfering with the effects of leucine on the pancreatic translational machinery, we studied the effect of leucine in vivo in diabetic rats and the effect of all 3 BCAA in preparations of isolated pancreatic acini also from diabetic rats. All BCAA stimulated 4E-BP1 and S6 phosphorylation independently of an increase in plasma insulin levels, as was described in skeletal muscle (12,32,48). It is noteworthy to highlight that BCAA and especially leucine can have different effects in different tissues, although they usually involve anabolic mechanisms. In skeletal muscle and adipose tissue, for example, leucine can stimulate total protein synthesis without changes in the phosphorylation status of these 2 translation factors in the absence of insulin (13,49), but in liver and pancreas, there is no change in protein synthesis.
Hyperphosphorylation of 4E-BP1 and S6K by amino acids, and leucine in particular, involves a signaling pathway that includes mTOR and is inhibited by rapamycin (15). Similar to what was described in rat skeletal muscle (8), rapamycin completely inhibited the phosphorylation of 4E-BP1 and S6 (used as a read-out of S6K activity) induced by leucine in the pancreas of normal rats, demonstrating that mTOR is upstream of both translation factors and is involved in the regulation of leucine stimulation of pancreatic translation.
How amino acids activate mTOR in acinar cells is unknown. In other cells, it was suggested that amino acids may activate the mTOR/Raptor/G-protein-ß-subunit-like protein complex by promoting phosphorylation of the tumor suppressor complex and inhibition of the small Ras homolog enriched in brain GTPase (50,51). Amino acids were also reported to activate mTOR through the inactivation of the AMP-activated protein kinase, when mitochondrial ATP is generated after the metabolism of amino acids in the mitochondria (40). Further studies are required to assess all of these mechanisms.
It is thought that amino acids do not activate protein kinases upstream of mTOR in contrast to growth-promoting hormones such as insulin and insulin-like growth factor (IGF)-1 (52). The stimulation of mTOR by insulin or IGF-1 may be mediated in part by phosphorylation of PKB/Akt on Ser 473, which results in its activation. Akt subsequently phosphorylates a residue (Ser 2448) on mTOR within a domain that normally acts to repress mTOR protein kinase activity (52). Amino acids seem to stimulate the translational machinery independently of Akt in rat (40,53–55) and human (56) skeletal muscle. In our study, however, Akt was activated by leucine in normal and diabetic rats. Moreover, all 3 BCAA stimulated Akt phosphorylation on Ser 473 in isolated acini from diabetic rats. The experiments in vitro also demonstrated that the effects do not require cholinergic stimulation. Although unexplored in the pancreas, a recent study (50) showing that BCAA activate a class III PI3K may provide a mechanism to explain the amino acid–induced activation of Akt.
In conclusion, it was demonstrated previously that stimulation of protein synthesis in the exocrine pancreas can be mediated by CCK, insulin, and the cholinergic analog, carbachol (21,24–26,28,29,31). The present study highlights the importance of dietary amino acids, especially the BCAA in the regulation of pancreatic protein synthesis at the translation/initiation level. BCAA stimulate the translational machinery of the exocrine pancreas independently of hormonal and neuronal input and may be "priming" the cell to synthesize a specific subgroup of proteins regulated by the mTOR pathway.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supplemental Figure 1 is available with the online posting of this paper at www.nutrition.org.
3 Supported by NIH grants DK-059578 to J.A.W. and DK-13499 to S.R.K.
5 Current address: Third Department of Internal Medicine, University of Occupational and Environmental Health, Japan, School of Medicine, Kitakyushu, Japan.
6 Abbreviations used: AU, arbitrary units; BW, body weight; CCK, cholecystokinin; 4E-BP1, eIF4E binding protein 1; eIF4E, initiation factor 4E; IGF, insulin-like growth factor; mTOR, mammalian target of rapamycin; PCA, perchloric acid; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; S6K, S6 kinase.
Manuscript received 1 February 2006. Initial review completed 21 February 2006. Revision accepted 12 April 2006.
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