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Departments of Cellular and Molecular Physiology, and Surgery, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
Address all correspondence and requests for reprints to: Charles H. Lang, Ph.D., Department Cellular and Molecular Physiology (H166), Pennsylvania State College of Medicine, Hershey, Pennsylvania 17033. E-mail: clang{at}psu.edu.
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Introduction |
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The elevation in plasma IGFBP-1 observed in various catabolic conditions appears to result primarily from increased synthesis and secretion of the protein by the liver (2, 3, 10, 11). However, where examined, the abundance of renal IGFBP-1 mRNA is concomitantly increased, and therefore the relative contribution of the kidney to the observed increase in plasma IGFBP-1 remains to be determined. Under nonstress conditions, the hepatic synthesis of IGFBP-1 is inversely proportional to the prevailing plasma insulin concentration (12). The ability of insulin to down-regulate hepatic IGFBP-1 synthesis is believed to be responsible for the daily fluctuations in plasma IGFBP-1 in response to fasting and refeeding (13). In contrast, increased concentrations of glucocorticoids and proinflammatory cytokines in the blood or tissues are predominant regulators of IGFBP-1 synthesis in catabolic conditions, and their effects on IGFBP-1 expression are mediated by enhanced transcription (2, 10, 14, 15).
Despite our expanding knowledge regarding the mechanisms by which IGFBP-1 synthesis is regulated, comparatively little is known regarding the physiological importance of increases in IGFBP-1 in catabolic conditions. Early studies reported that incubation of various cell types with IGFBP-1 generally inhibits a wide range of IGF-I actions in vitro (13). Of particular interest are studies reporting that IGFBP-1 decreases the ability of IGF-I to stimulate glucose uptake and protein synthesis in cultured human muscle cells (16) because such metabolic defects might be causally related to the development of muscle wasting during prolonged catabolic conditions (17, 18). More recently, the development of transgenic mice that constitutively overexpress IGFBP-1 have been used to address the paucity of information regarding the in vivo actions of IGFBP-1. These transgenic mice have decreased body weight and impaired glucose homeostasis, the latter evidenced by fasting hyperglycemia and hyperinsulinemia as well as the development of insulin resistance in muscle (19, 20, 21). Again, these metabolic derangements are also characteristic of many catabolic conditions (22). However, the impact of elevated IGFBP-1 on muscle protein balance per se has not been reported. Although the availability of IGFBP-1-overexpressing mice represents a novel and important tool for elucidation of the in vivo effects of IGFBP-1, interpretation of data from these animals is complicated by both known and unknown compensatory changes that occur during prenatal and postnatal development.
The purpose of the present study was to determine whether acutely increasing the circulating concentration of IGFBP-1 to levels observed in catabolic conditions would alter muscle protein balance in rats. The experiments performed address the hypothesis that acute elevations in plasma IGFBP-1 will decrease the circulating concentration of free IGF-I and thereby impair rates of muscle protein synthesis in otherwise normal rats. Because of the temporal association between increases in IGFBP-1 and the erosion of lean body mass in catabolic conditions, these data will provide additional support for the physiological relevance of stress-induced increases in IGFBP-1.
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Materials and Methods |
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For all experiments, rats were anesthetized with an ip injection of ketamine and xylazine (90 and 9 mg/kg, respectively), and sterile surgery was performed to implant catheters in the carotid artery and jugular vein, as previously described (2, 9). After surgery, rats were housed individually, provided food and water ad libitum, and allowed to recover for 3 d. At this time point, the catheterized rats were indistinguishable from naive control animals, in that there were no differences in food or water consumption or the plasma concentration of total IGF-I or IGFBP-1 (data not shown).
Tissue protein synthesis and RNA content
For the first experimental series, conscious unrestrained rats were infused with human IGFBP-1 purified from amniotic fluid or vehicle. IGFBP-1 (Calbiochem, La Jolla, CA) was diluted in 0.1% human serum albumin and administered as a primed, constant iv infusion (100 µg/kg and 100 µg/kg·h) at a rate of 1 ml/h. Time-matched control animals were infused with an equal volume of vehicle. Serial arterial blood samples (350 µl each) were collected into heparinized syringes at selected time points thereafter, and plasma was used for the determination of various metabolic parameters, as described below. Blood volume was replaced with an equal volume of sterile 0.9% saline. Preceding each blood sample, the mean arterial blood pressure and heart rate were determined by a pressure transducer attached to the arterial catheter (model 79E, Grass Instrument Co., Quincy, MA).
Four hours after the start of the IGFBP-1 infusion, the in vivo rate of tissue protein synthesis was determined using the flooding dose technique, as originally described by Garlick et al. (23) and modified by our laboratory (24). Animals were injected iv with L-[2,3,4,5,6-3H]phenylalanine ([3H]Phe) (150 mM; 30 µCi/ml; 1 ml/100 g body weight). Ten minutes later, a blood sample was collected from the arterial catheter into a heparinized syringe. The gastrocnemius, soleus, heart (ventricle only), liver, and kidney were rapidly excised and frozen between aluminum blocks precooled to the temperature of liquid nitrogen. The frozen tissues were powdered under liquid nitrogen using a mortar and pestle. Blood samples were centrifuged (13,000 x g for 1 min at 4 C), and plasma was collected. Tissue and plasma samples were stored at -70 C until analyzed. A portion of the powdered tissue was homogenized in ice-cold perchloric acid (PCA), and the supernatant was used to estimate the rate of incorporation of [3H]Phe into protein (24). The specific radioactivity was calculated by dividing the amount of radioactivity in the peak corresponding to Phe by the concentration of the amino acid in the same fraction. Rates of protein synthesis were calculated as previously described (24) using the mean plasma Phe specific activity as the precursor pool.
Total RNA was measured in homogenates of gastrocnemius. Briefly, frozen powdered tissue was homogenized in 5 vol ice-cold 10% trichloroacetic acid. After centrifugation, the supernatant was discarded, and the remaining pellet was mixed with 6% PCA. The sample was centrifuged at 10,000 x g for 6 min at 4 C. The supernatant was discarded, and the procedure was repeated. To the pellet, 0.3 N KOH was added, and the samples were placed in a 50 C water bath for 1 h. Samples were then mixed with 4 N PCA and centrifuged. The concentration of RNA in the supernatant was determined by measuring the absorbance at 260 nm and correcting for the absorbance at 232 nm. These data were used to calculate translational efficiency, which equals the rate of protein synthesis for a particular tissue divided by the RNA content for that tissue.
Glucose kinetics
For the second series of experiments, animals were surgically prepared as described above, except that rats were fasted overnight to minimize glucose derived from hepatic glycogen and gastrointestinal absorption as a contributor to the determined rate of glucose turnover. All experiments were performed the next morning starting at approximately 0800 h on conscious, unrestrained rats. A primed, constant iv infusion of [3-3H]glucose (HPLC purified; specific activity, 13.5 Ci/mmol; Du Pont-NEN Life Science Products, Boston, MA) was started to determine basal glucose kinetics. A 5-µCi bolus injection of labeled glucose was administered, followed by the infusion of tracer at a rate of 0.83 µCi/min that was continued for the remainder of the experimental protocol (25). Two baseline arterial blood samples were collected at 120 and 140 min (350 µl each). Thereafter, rats were infused with human IGFBP-1 (100 µg/kg and 100 µg/kg·h) or an equal volume of vehicle, as described above. Additional blood samples were obtained 30, 60, 120, 180, and 240 min later. Animals received 0.5 ml sterile saline after each blood sample to maintain blood volume. The plasma glucose, lactate, and insulin concentrations as well as glucose specific activity were determined on each sample. The plasma glucose and lactate concentrations were determined using a rapid analyzer (GL5, Analox Instruments, Lunenbrug, MA). Glucose specific activity was determined on neutralized supernatants of deproteinized plasma, as previously described (25). An aliquot of the supernatant was used to determine [3H]glucose specific activity after tritiated water was evaporated. The rate of glucose appearance (Ra) and glucose disappearance (Rd) were calculated using nonsteady state equations (26).
IGF-I determination
The concentration of total IGF-I in plasma was determined using a modified acid-ethanol (0.25 N HCl/87.5% ethanol) procedure with cyroprecipitation. This method has been previously demonstrated to remove essentially all IGFBP-1 from plasma even under conditions where the concentration of IGFBP-1 is markedly elevated (6). Samples for total IGF-I were analyzed in duplicate by RIA, and assay characteristics have been previously described (2, 3, 4, 5, 6, 7). Previous studies have demonstrated that a 10-fold excess of IGFBP-1 does not interfere with the assay for total IGF-I (6). The plasma concentration of free (or readily dissociable) IGF-I was determined by centrifugal ultrafiltration as originally described by Frystyk et al. (27) and modified by our laboratory (10). Briefly, the plasma samples were diluted 1:5 with Hanks’ Balanced Salt Solution (pH 7.4; with 5% BSA) and prefiltered through a 0.22-µm pore size filter (Millex-GV, Millipore, Molsheim, France) to remove debris. The prefiltered samples were then added to Amicon YMT 30 membranes and MPS-1 supporting devices (Amicon Division, W. R. Grace Co., Beverly, MA) and centrifuged at 300 x g (1500 rpm) at 37 C for 100 min. The ultrafiltrate was collected from 40–100 min of centrifugation and used for the IGF-I RIA. All samples for free IGF-I were analyzed in the same assay. Preliminary studies indicated that the addition of human IGFBP-1 to control rat plasma samples for 60 min before ultrafiltration dose-dependently decreased the measured concentration of free IGF-I (data not shown).
IGFBP-1 binding assay
IGFBP-1 binding was characterized with a solid phase microtiter plate assay. IGF-I (Genentech, South San Francisco, CA) was adsorbed to a 96-well MaxiSorp microtiter plate (Nalge Nunc International, Rochester, NY) in carbonate buffer (20 mM, pH 9.5) overnight. Nonspecific binding sites were blocked with assay diluent (PharMingen, San Diego, CA), and IGFBP-1 binding to IGF-I on the plate was detected with an excess of antihuman IGFBP-1 polyclonal antibody (Upstate Biotechnology, Inc., Lake Placid, NY). IGFBP-1 antibody complexes were detected with a horseradish peroxidase-conjugated goat antirabbit immunoglobulin and incubation with the colorimetric substrate tetramethylbenzidine (PharMingen). The peroxidase reaction was stopped with 2 N H2SO4, and the absorbance in each well was read at 450 nm. IGF-I binding to the microtiter plate and IGF-I·IGFBP-1 complex formation were linear. IGFBP-1 binding was dependent upon the amount of IGF-I bound to the plate and was linear up to a 1:1 molar ratio, at which point IGFBP-1 binding was saturated. For competition binding experiments IGF-I peptides had the following order of potencies: IGF-I > IGF-II > des IGF-I > insulin. IGF peptides were preincubated with IGFBP-1 for 1 h at room temperature and then incubated with the microtiter plate at 4 C overnight. For the IGFBP-1 heat inactivation experiment, 2 µg/ml IGFBP-1 (Calbiochem, La Jolla, CA) were heated at 100 C for 5–60 min. Duplicate samples from each time point were assayed for IGF-I binding.
RNA extraction and Northern blotting
Total RNA was isolated using TRI Reagent TR-118 as outlined by the manufacturer (Molecular Research Center, Cincinnati, OH). Samples of total RNA (20–100 µg) were electrophoresed under denaturing conditions in 1% agarose/6% formaldehyde gels, as described previously (3, 10). The running buffer was 1x HEPES. Northern blotting occurred via capillary transfer to 
-Probe GT blotting membranes (Bio-Rad Laboratories, Hercules, CA). An 800-bp probe from rat IGF-I (Peter Rotwein, St. Louis, MO) and a 407-bp probe from IGFBP-1 were labeled using a Random Primed DNA Labeling kit (Roche, Indianapolis, IN). A rat 18S oligonucleotide, synthesized by the Macromolecular Core Facility at Pennsylvania State College of Medicine using a Perseptive Biosystems Expedite 8909 Nucleic Acid Synthesizer, was used for normalization of RNA loading. The oligonucleotide was radioactively end-labeled using T4 polynucleotide kinase (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were prehybridized and hybridized at 42 C in 50% formamide/6x SSPE [3.6 M NaCl, 0.2 M Na phosphate, 0.02 EDTA (pH 7.7)]/5x Denhardt’s/1% sodium dodecyl sulfate/10% dextran sulfate/herring testis DNA (100 µg/ml). All membranes were washed at room temperature twice in 2x standard saline citrate (SSC)/0.1% SDS for 5 min and once in 0.1x SSC/0.1% SDS for 15 min. Additionally, membranes hybridized with rat IGF-I were washed at 55 C in 0.1x SSC/0.1% SDS for 15–30 min. Finally, membranes were exposed to a phosphorimager screen, and the resultant data were analyzed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Data were normalized to the content of ribosomal 18S RNA. Relative mRNA abundance was expressed as the ratio between the particular mRNA and 18S mRNA. This ratio was arbitrarily set at 1.0 for tissues from control animals. RNA samples from tissues of rats infused with vehicle or IGFBP-1 were electrophoresed on the same gel.
Western blotting
Plasma samples were separated on a 12.5% SDS-PAGE gel under nonreducing conditions as previously described (3, 10). Separated proteins were electroblotted onto nitrocellulose and blocked for 2 h at room temperature with Tris-buffered saline containing 1% nonfat dry milk. The membranes were then incubated with antiserum against rat IGFBP-1 (Upstate Biotechnology, Inc.) at room temperature for 2 h. Antigen-antibody complexes were identified with goat antirabbit immunoglobulin G tagged with horseradish peroxidase (Sigma-Aldrich Corp., St. Louis, MO) and exposed to the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) for 1 min and to x-ray film for 10–30 sec. Bands were scanned (Microtek ScanMaker IV, Carson, CA) and analyzed using NIH Image 1.6 software. Samples from both groups were run on the same gel, and the data were expressed as a percentage of the control value.
Distal components of signaling pathways important for the translational control of protein synthesis were also assessed in gastrocnemius. The tissue preparation was essentially the same as previously described (28, 29). Briefly, fresh muscle homogenates were prepared in a 1:7 ratio of ice-cold homogenization buffer [20 mM HEPES (pH 7.4), 2 mM EGTA, 50 mM NaF, 100 mM KCl, 0.2 mM EDTA, 50 mM ß-glycerophosphate, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonylfluoride, 1 mM benzamidine, and 0.5 mM sodium vanadate] using a Polytron homogenizer and were centrifuged at 10,000 x g for 10 min. The supernatant was aliquoted into microcentrifuge tubes, and 2x sample buffer [2 ml 0.5 M Tris (pH 6.8), 2 ml glycerol, 2 ml of 10% sodium dodecyl sulfate, 0.2 ml ß-mercaptoethanol, 0.4 ml of a 4% solution of bromophenol blue, and 1.4 ml water to a final volume of 8 ml] was added in a 1:1 ratio. The samples were boiled for 5 min and cooled on ice before being used for Western blot analysis. The samples were subjected to electrophoresis on a 7.5% polyacrylamide gel for S6K1 (ribosomal protein S6 kinase 1). Proteins were electrophoretically transferred to nitrocellulose membranes. The blots were incubated with antirabbit total S6K1. The blots were developed as described above. The 4E-BP1·eukaryotic initiation factor-4E (eIF4E) and eIF4G·eIF4E complexes were quantified as previously described (28, 29). Briefly, eIF4E was immunoprecipitated from aliquots of 10,000 x g supernatants using an anti-eIF4E monoclonal antibody (Drs. Jefferson and Kimball, Hershey, PA). The antibody-antigen complex was collected using magnetic beads and subjected to electrophoresis using a 7.5% or 15% polyacrylamide gel. Proteins were then electrophoretically transferred to a nitrocellulose membrane. The blots were incubated with a mouse antihuman eIF4E antibody, a rabbit antirat 4E-BP1 antibody, or a rabbit anti-eIF4G antibody for 1 h at room temperature. The phosphorylated forms of 4E-BP1 were measured after immunoprecipitation of 4E-BP1 from the tissue homogenates after centrifugation at 10,000 x g. The various phosphorylated forms of 4E-BP1 were separated by SDS-PAGE and analyzed by protein immunoblotting. The blots were then developed with enhanced chemiluminescence, and the autoradiographs were scanned for analysis as described above.
Epitrochlearis muscle incubation
Epitrochlaris muscles were incubated in vitro as described previously (30, 31). This muscle was selected because numerous studies have demonstrated that catabolic states preferentially affect protein metabolism in muscles composed of mixed fast-twitch fibers (32). On the day of the experiment, rats were anesthetized with pentobarbital, and the skin on each of the forearms was removed. The epitrochlearis muscles were excised intact and immediately placed in Krebs-Henseleit bicarbonate buffer. The muscles were quickly rinsed and transferred to vials containing 2 ml buffer. The vials were capped and immediately oxygenated. One muscle from each rat was incubated under basal conditions, and the other muscle was incubated in the presence of IGFBP-1 and/or IGF-I. Muscles were preincubated for 30 min, after which they were transferred to fresh medium and incubated for an additional 3 h, with a change of buffer every 60 min. During the final 60 min, the buffer was supplemented with 1.2 mM L-[14C]Phe (0.15 µCi/ml). At the end of the incubation, muscles were removed from the buffer, trimmed of connective tissue, immersed in ice-cold trichloroacetic acid, and weighed. The incubation medium was frozen and stored at -20 C for analysis of tyrosine and the specific radioactivity of phenylalanine.
The Krebs-Henseleit bicarbonate buffer consisted of 120 mM NaCl, 4.8 mM KCl, 25 mM NaHCO3, 2.5 mM CaCl2, 1.2 mM KH2PO4, and 1.2 mM MgSO4 (pH 7.4) supplemented with 5 mM glucose, 5 mM HEPES, 0.1% (wt/vol) BSA, 0.17 mM leucine, 0.20 mM valine, and 0.10 mM isoleucine. Muscles were incubated at 37 C under an atmosphere of 95% O2-5%CO2. In some experiments IGFBP-1 (1000 ng/ml) and/or IGF-I (20 ng/ml) was added. The rate of protein synthesis was calculated using the amount of radioactivity incorporated into muscle protein over a 1-h period divided by the specific radioactivity of Phe in the incubation medium.
Other assays
The plasma concentration of human IGFBP-1 in rats was assayed using an immunoradiometric assay that does not cross-react with rat IGFBP-1 (Diagnostic Systems Laboratories, Webster, TX). The plasma insulin concentration was determined using a rat-specific RIA (Linco Research, Inc., Louis, MO). Plasma corticosterone concentrations were also determined by RIA (Diagnostic Products, Los Angeles, CA). Aspartate aminotransferase activity in plasma was determined, as an index of hepatic toxicity, using a standard enzymatic assay (Sigma-Aldrich Corp.). The ATP and creatine phosphate (CP) contents of gastrocnemius were used as an assessment of the energy status of the muscle. An aliquot of powdered tissue was extracted in cold PCA, neutralized, and used to determine ATP and CP by standard fluorometric methods.
Statistics
For in vivo studies, data were obtained from two separate experimental series, each containing control and IGFBP1-infused rats (n = 8 and n = 9, respectively). Experimental values are presented as the mean ± SE. Data were analyzed using t test to determine the treatment effect. For the in vitro studies (n = 12–27 muscles/group), statistical evaluation of all four groups was performed using ANOVA to test for overall differences among groups, followed by Student-Newman-Keuls test to determine the treatment effect. When heterogeneity of variance was detected, data were transformed to logarithmic values before statistical analysis (Instat, San Diego, CA). Statistical significance was set at P < 0.05.
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Results |
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). At a fixed amount of IGF-I (25 ng/well or 
2 x 1012 molecules) IGFBP-1 became saturated with IGF-I when the molar ratio approached 1:1 (88 ng/well IGFBP-1 or 2 x 1012 molecules; Fig. 1B). Binding in the expected molar ratio suggests that the IGFBP-1 preparation infused was not significantly contaminated with IGF-I or IGF-II. When IGF-I was added to IGFBP-1 in solution, it competed with IGF-I on the plate for IGFBP-1 binding with a Ka of approximately 1 x 109 M (Fig. 1C). In contrast, up to 100-fold more des-IGF-I did not compete for IGFBP-1 binding. IGFBP-1 purified from amniotic fluid has previously been shown to contain a mixture of nonphosphorylated and phosphorylated IGFBP-1 that has an affinity for IGF-I in the concentration range found here. In addition, the IGFBP-1 preparation used herein has binding characteristics similar to those of IGFBP-1 secreted from the HepG2 human hepatoma cell line (Frost, R. A., and C. H. Lang, unpublished observation). Finally, IGFBP-1 binding was heat labile. The IGF-binding activity of IGFBP-1 was progressively impaired by longer periods of IGFBP-1 heat inactivation. Boiling of IGFBP-1 for 20 min was sufficient to completely eliminate IGF-binding activity (Fig. 1D).
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illustrates that each of the three doses examined incrementally increased the plasma concentration of human IGFBP-1. In addition, a steady state plasma concentration was achieved between 2 and 3 h of the IGFBP-1 infusion. Values for the last 0.5 h of the infusion protocol averaged 5 ± 2, 33 ± 6, and 71 ± 8 ng/ml, respectively, for the three groups. There was no detectable human IGFBP-1 in the plasma of time-matched control rats infused with an equal volume of vehicle. Based on these observations, a primed, constant infusion rate of 100 µg/kg and 100 µg/kg·h for 4 h was used for all subsequent experiments.
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, top). In contrast, the concentration of free IGF-I in the blood was decreased by 25% as early as 1 h after the start of IGFBP-1 and was reduced 53–65% during the last 2 h of the experimental protocol (Fig. 3, bottom).
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, B and C). The lack of significant cross-reactivity of the antirat IGFBP-1 antibody toward human IGFBP-1 is illustrated in Fig. 4A.
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, the infusion of human IGFBP-1 increased rat IGFBP-1 mRNA in both liver and kidney between 2- and 3-fold.
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, there was significant 40% reduction in IGF-I mRNA content in liver from IGFBP-1-infused rats compared with values from time-matched control animals. The IGF-I mRNA content in the kidney and gastrocnemius also tended to decline (15–20%) after the infusion of IGFBP-1, but these changes failed to reach statistical significance.
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illustrates that after a 4-h infusion of IGFBP-1 the in vivo rate of protein synthesis was decreased in the predominantly fast-twitch gastrocnemius muscle by 20%. This reduction was observed regardless of whether the protein synthesis rate was normalized to wet weight of tissue (Fig. 7A) or per protein content (data not shown). In contrast, no significant change in the in vivo protein synthetic rate was detected for soleus muscle, heart, liver, or kidney (Table 1).
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, middle panel). As a result of these changes, the efficiency of translation was also decreased in muscle by 20% (Fig. 7, bottom panel).
S6K1, also referred to as p70 S6 kinase, is a downstream serine/threonine kinase in the insulin/IGF-I signaling cascade and is activated by phosphorylation on at least seven different serine/threonine resides (33). When the kinase is subjected to SDS-PAGE, it resolves into multiple bands with different electrophoretic mobilities dependent on the extent of phosphorylation at these different serine/threonine sites. In this regard, the most slowly migrating forms represent the heavily phosphorylated and thus the highly active form of the kinase (34). There was a basal level of S6K1 phosphorylation in muscle from control rats that was resolved into at least three separate bands (Fig. 8A, arrows). In muscles from rats infused with IGFBP-1 there was an increased mobility of the top band, indicating a relative dephosphorylation of S6K1.
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) or the distribution of this initiation factor, as evidenced by the unchanged amounts of eIF4E associated with eIF4G and 4E-BP1 (Fig. 8, B and C, respectively). Finally, elevations in IGFBP-1 did not alter the phosphorylation of 4E-BP1 (Fig. 8E), compared with muscle from control animals. There was no difference in the ATP concentration in gastrocnemius between control (6.82 ± 0.16 µmol/g wet weight) and IGFBP-1-infused rats (7.03 ± 0.19 µmol/g wet weight). Likewise, there was no difference in CP levels between the two groups (20.6 ± 1.7 and 19.5 ± 1.8 µmol/g wet weight, respectively). These data suggest that a generalized energy deficit is not responsible for the decrease in muscle protein synthesis observed in rats infused with IGFBP-1.
The alterations in muscle protein synthesis as well as the increase in endogenous IGFBP-1 might be an indirect effect of changes in other hormones or a generalized stress response to the infusion of IGFBP-1. However, IGFBP-1 did not significantly alter the plasma concentration of either insulin or corticosterone during the experimental protocol (Table 2). In addition, mean arterial blood pressure and heart rate were not altered in IGFBP-1-infused rats at the time points assessed (Table 2). Finally, there was no difference in the plasma aspartate aminotransferase levels between IGFBP-1-infused rats and time-matched control animals, suggesting that the alterations in muscle protein synthesis and select components of the IGF system were not mediated secondarily via a generalized hepatotoxicity.
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illustrates that incubation of the epitrochlearis muscle for 3 h with IGF-I increased protein synthesis by 35%. Conversely, incubation of muscles with IGFBP-1 decreased the synthetic rate by 23% under basal conditions. Furthermore, IGFBP-1 completely prevented the IGF-I-induced increase in protein synthesis.
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) or the plasma insulin concentrations (data not shown) between rats infused with IGFBP-1 and those administered vehicle. Moreover, isotope dilution-determined rates of basal hepatic glucose production (glucose Ra) and peripheral glucose uptake (glucose Rd) were also not different between groups during the 4-h infusion protocol.
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, top panel). However, there was no increase in the hepatic IGFBP-1 mRNA content in rats infused with heat-inactivated IGFBP-1 compared with saline-infused rats. Likewise, the decrease in the plasma free IGF-I concentration produced by the infusion of native IGFBP-1 was not observed in rats administered heat-inactivated IGFBP-1 (Fig. 10, bottom panel). Rats injected iv with native or heat-treated endotoxin (Escherichia coli, Difco, Detroit, MI; 10 µg/kg BW) had essentially an identical increase in hepatic IGFBP-1 mRNA and a decrease in the plasma free IGF-I concentration (Fig. 10). Because endotoxin is relatively heat stable, and IGFBP-1 is heat labile, these data suggest that the observed changes produced by the infusion of IGFBP-1 were not due to endotoxin present in the infusate. This conclusion is supported by our above-mentioned data demonstrating that the IGFBP-1 infusion did not alter the plasma concentration of glucose or corticosterone, two relatively sensitive end points of endotoxin action (2).
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The large majority of IGF-I present in the circulation is restricted to the intravascular compartment because of its binding to IGFBP-3 and the acid-labile subunit. Lesser amounts of IGF-I bind to one of several smaller IGFBPs, forming binary complexes. Hence, normally only a relatively small fraction of the total IGF-I circulates in the free, unbound form. The concentration of free IGF-I is believed to represent the peptide that is bioavailable and is a primary determinant of the tissue response to IGF-I (39, 40). In general, when present in molar excess, IGFBP-1 inhibits ligand-receptor interaction and attenuates or completely prevents IGF-I bioactivity (16). Conversely, when sequestered IGF-I is released from IGFBP-1 it becomes metabolically active and capable of activating the type 1 IGF receptor (41). The increased IGFBP-1 observed in various catabolic conditions is believed to contribute to the concomitant decrease in free IGF-I (17). In the current study the infusion of IGFBP-1 reduced the plasma concentration of free IGF-I by more than 50% within 4 h. Moreover, this reduction was accomplished without a significant reduction in the total IGF-I concentration. Therefore, this experimental condition is similar to others where there is a disproportionate decrease in free IGF-I relative to the decline in the total IGF-I concentration (9, 42, 43). Consequently, the observed metabolic and endocrine changes produced by the elevation of IGFBP-1 are independent of changes in the plasma concentration of total IGF-I and may be due to either a decreased availability of free IGF-I and/or the IGF-independent changes induced by the elevation in IGFBP-1 per se.
Elevations in IGFBP-1 are known to influence glucose metabolism. For example, IGFBP-1 transgenic mice demonstrate fasting hyperglycemia and hyperinsulinemia as well as glucose intolerance that are largely attributable to defects in pancreatic insulin secretion (19, 20, 36). Furthermore, the bolus injection of IGFBP-1 purified from human amniotic fluid produces transient hyperglycemia in rats (44) that may in part be due to the suppression of muscle glucose uptake mediated by IGF-I (45). In contrast, in the present study the acute elevation of plasma IGFBP-1 did not alter basal glucose homeostasis, as evidenced by the similar plasma concentrations of glucose, lactate, and insulin. Furthermore, we were unable to detect a difference in glucose Ra and glucose Rd between control and IGFBP-1-infused rats, suggesting that elevations in IGFBP-1 did not alter either the rate of hepatic glucose production or the rate of glucose uptake by peripheral tissues under basal fasted conditions. The IGFBP-1 infusion also did not alter the hepatic mRNA content of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (Lang, C. H., and R. A. Frost, unpublished observation), a finding consistent with the unchanged rate of glucose production. The apparent discrepancy between our results and those of Lewitt et al. (44, 45) might be due to the injection of 4- to 5-fold more IGFBP-1 and the resultant approximately 10-fold greater elevation in the plasma IGFBP-1 concentration in the later study. The plasma IGFBP-1 concentrations achieved in this previous study appear more pharmacological, whereas the concentration produced in the current study is more pathophysiological (4, 7, 37, 38). Collectively, these data suggest that the disturbance in basal glucose homeostasis that is characteristic of many catabolic conditions is unlikely to be mediated by the acute elevation of IGFBP-1; however, sustained elevations in IGFBP-1 may well contribute to the glucose dyshomeostasis observed in more prolonged catabolic states, such as sepsis, diabetes, human immunodeficiency virus infection and chronic alcohol abuse.
The elevation of plasma IGFBP-1 was associated with a decreased rate of total protein synthesis in the gastrocnemius muscle. The ability of increased IGFBP-1 or decreased free IGF-I to impair protein synthesis appears restricted to fast-twitch skeletal muscle, because no change in synthetic rate was detected in the slow-twitch soleus muscle or cardiac muscle. These results are similar to those reported in other catabolic conditions where muscles with a predominance of fast-twitch fibers demonstrate a greater susceptibility to decrements in protein synthesis (8, 32, 46). Moreover, these findings are consistent with previous studies indicating that elevations in plasma IGF-I predominantly increase in vivo protein synthesis in gastrocnemius, with only modest changes in soleus and heart and no change in the synthetic rate of many peripheral organs (47). Finally, these findings also suggest that skeletal muscle protein synthesis is a more sensitive indicator of IGFBP-1 excess and decreases in free IGF-I than alterations in carbohydrate metabolism.
IGFBP-1 can be secreted as a phosphoprotein. The phosphorylated isoforms often exhibit enhanced affinity for IGF peptides compared with the nonphosphorylated form, and this posttranslational modification may increase its ability to inhibit various IGF-I actions (48). The IGFBP-1 used in the current study was isolated from amniotic fluid and has been previously shown to contain a mixture of nonphosphorylated and phosphorylated IGFBP-1 isoforms (49). Therefore, an increase in the concentration of the phospho-variants might be expected to have a relatively greater inhibitory effect on muscle protein synthesis. This concern may be particularly relevant because our previous studies have demonstrated that essentially all of the trauma-induced increase in plasma IGFBP-1 in humans exists in the highly phosphorylated isoforms (5).
Subsequent analyses have begun to reveal the mechanism by which IGFBP-1 decreases protein synthesis in the gastrocnemius. The IGFBP-1 infusion did not alter the total RNA content of the gastrocnemius. These data suggest that the decreased protein synthesis did not result from alterations in the relative abundance of ribosomes, but instead was due to an impairment in translational efficiency (18). IGF-I stimulates protein synthesis via increases in translational efficiency (28), and conversely, defects in translational efficiency have been previously reported in a number of catabolic conditions associated with the erosion of lean body mass (28, 29, 50). Anabolic signals are propagated to the translational apparatus using mammalian target of rapamycin (mTOR) as an intermediate (34). Several lines of evidence suggest that mTOR is a common upstream activator of both S6K1 and 4E-BP1. Furthermore, these two metabolic regulators appear to lie on parallel pathways (34). The enzymatic activity of S6K1 is regulated by phosphorylation and is pivotal for the maintenance of normal rates of protein synthesis. S6K1, in turn, phosphorylates the 40S ribosomal protein S6, thereby regulating the selective translation of mRNAs containing a tract of oligopyrimidines at their 5'-transcriptional start site that encode for components of the translational apparatus. Our data clearly demonstrate a decreased phosphorylation of S6K1 in muscle from rats infused with IGFBP-1. In contrast, we could not detect any significant difference in the relative phosphorylation of 4E-BP1 or any significant alterations in the distribution of eIF4E between the active eIF4E·eIF4G complex and the inactive 4E-BP1·eIF4E complex. These data suggest that an IGFBP-1-induced decrease in S6K1 phosphorylation is responsible at least in part for the concomitant impairment in muscle protein synthesis, but that an inhibition of mTOR activity is unlikely to be solely responsible for the defect in S6K1 phosphorylation.
The decrease in protein synthesis and translational efficiency could not be explained by a generalized decline in the muscle content of high energy phosphates (ATP and CP) or a decrease in the prevailing concentration of the anabolic hormone insulin. Furthermore, the infusion of IGFBP-1 did not increase the plasma corticosterone concentration. Such an increase would be indicative of a generalized stress response and provide a possible mechanism for the reduction in translation initiation (46, 50). The ability of elevated concentrations of IGFBP-1 to impair basal and the IGF-I-induced increase in muscle protein synthesis in the incubated epitrochlearis muscle, another fast-twitch muscle, further suggests that the effect of IGFBP-1 is either direct or via a reduction in tissue IGF-I content. These data also suggest that the inhibitory effect of IGFBP-1 on muscle protein synthesis did not result from the production of some secondary mediator (e.g. hormone or cytokine), and they minimize endotoxin contamination as a potential mechanism, because incubated skeletal muscles are unresponsive to high doses of this immunomodulator (51). Finally, because of the inability of exogenous IGF-I to stimulate protein synthesis in peripheral organs (47), it was not totally unexpected that protein synthetic rates in liver and kidney were unaltered under conditions where IGFBP-1 was increased and free IGF-I was decreased.
The infusion of IGFBP-1 also produced several rather unexpected results. Western blot analysis with a rat-specific antibody indicated that the infusion of human IGFBP-1 produced a several-fold increase in the plasma concentration of rat IGFBP-1. Moreover, this increase appeared to result from an increased rate of IGFBP-1 synthesis, as the mRNA content for this binding protein was increased in both liver and kidney. Although increases in glucocorticoids and decreases in insulin are known to up-regulate IGFBP-1 synthesis (52), these hormonal regulators do not appear to be operational in the current experimental paradigm. Furthermore, the increased IGFBP-1 is unlikely to be mediated indirectly via a change in free IGF-I because this growth factor has not been shown to affect IGFBP-1 expression in primary rat hepatocytes (53). Alternatively, this response may represent a direct effect of IGFBP-1 mediated by the interaction of its Arg-Gly-Asp (RGD) sequence with various cell membrane integrin receptors (16).
Based on reports demonstrating that increases in IGF-I suppress the IGF-I mRNA content in liver and muscle (54, 55), we anticipated that a decrease in the free IGF-I concentration would increase the IGF-I mRNA content in rats infused with IGFBP-1. However, in the present study the steady state IGF-I mRNA content was significantly decreased in liver and also tended to be reduced (15–20%) in kidney and gastrocnemius of rats infused with IGFBP-1. A reduction in the plasma IGF-I concentration has been reported to occur in transgenic mice with liver-specific expression of the human IGFBP-1 (56). The mechanism by which the acute elevation in IGFBP-1 down-regulates IGF-I mRNA in liver, and possibly other tissues, is unknown but does not appear to result from a generalized hepatotoxic effect of IGFBP-1 (e.g. no increase in plasma ALT levels) or the overall suppression of hepatic gene transcription (e.g. no change in phosphoenolpyruvate carboxykinase mRNA and increases in IGFBP-1 mRNA content). Hence, this ability of IGFBP-1 to negatively regulate IGF-I mRNA may potentially contribute to the decreased IGF-I peptide concentration in plasma and tissues and, in turn, may negatively impact tissue protein balance in sustained models of catabolic stress.
In summary, our results indicate that exogenous administration of human IGFBP-1 to rats dose-dependently increases circulating levels of IGFBP-1 and markedly reduces the prevailing plasma concentration of free IGF-I. As a result of these changes, the basal rate of protein synthesis is selectively decreased in fast-twitch skeletal muscle via an impairment of translational efficiency at the level of S6K1. In contrast, basal rates of glucose production and disposal were not altered, indicating that basal whole body glucose turnover is relatively insensitive to acute pathophysiological elevations in plasma IGFBP-1. In addition, the content of IGFBP-1 mRNA is increased and the content of IGF-I mRNA decreased in liver from rats infused with IGFBP-1. None of the above-mentioned changes could be attributed to alterations in the circulating concentrations of insulin, total IGF-I, or corticosterone. Collectively, these data suggest that the acute elevations in IGFBP-1 observed in response to various catabolic insults can negatively impact muscle protein balance by decreasing the plasma concentration of free IGF-I and protein synthesis as well as by further enhancing the hepatic synthesis of IGFBP-1.
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Abbreviations: CP, Creatine phosphate; eIF, eukaryotic initiation factor; IGFBP, IGF-binding protein; mTOR, mammalian target of rapamycin; PCA, perchloric acid; Phe, phenylalanine; Ra, rate of glucose appearance; Rd, rate of glucose disappearance; SSC, standard saline citrate.
Received December 24, 2002.
Accepted for publication May 28, 2003.
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. Growth Horm IGF Res 11:25–260
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), 40 (
), or 100 µg/kg·h (
) of IGFBP-1. IGFBP-1 was determined by a human-specific immunoradiometric assay. Time-matched control rats were infused with an equal volume of vehicle (1 ml/h), and plasma samples yielded no detectable human IGFBP-1. Values are the mean ± SE (n = 4/group).
, Values from rats infused with IGFBP-1; 
, values from time-matched control animals. Values are the mean ± SE (n = 8–9 rats/group at each time point). *, P < 0.05 compared with values from time-matched control rats.
-form. A total of seven muscle samples from each group were electrophoresed for each parameter. A, All samples from rats infused with IGFBP-1 clearly demonstrated a decrease in the intensity of the top band (e.g. top arrow). B–E, Data were quantified by densitometric analysis of immunoblots, and there were no significant differences (e.g. P > 0.05) between values from control and IGFBP-1-infused rats for these particular parameters.
