Departments of Cellular and Molecular Physiology, and Surgery, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033-0850
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous studies demonstrate that thermal injury decreases circulating levels of insulin growth factor I (IGF-I) and alters the plasma concentration of several IGF binding proteins (IGFBP), but the mechanisms for these alterations have not been elucidated. In the current study, a 30% total body surface area full-thickness scald burn was produced in anesthetized rats, and animals were studied 24 h later. The plasma concentration of both total and free IGF-I was decreased (38 and 65%, respectively) in burn rats compared with values from time-matched control animals. Thermal injury decreased the IGF-I peptide content in liver ~40%, as well as in fast-twitch skeletal muscle (56-69%) and heart (28%). In contrast, IGF-I content in kidney was elevated by 36% in burn rats. Northern blot analysis of liver indicated that burn decreased the expression of small (1.7- and 0.9- to 1.2-kb) IGF-I mRNA transcripts but increased the expression of the 7.5-kb transcript. In contrast, there was a coordinate decrease in all IGF-I mRNA transcripts in muscle and kidney of ~30%. For liver, muscle, and kidney, there was no significant difference in the expression of growth hormone receptor mRNA between control and burn rats. Thermal injury increased plasma IGFBP-1 levels, and this change was associated with increased IGFBP-1 mRNA in both liver and kidney. IGFBP-3 levels in plasma were concomitantly decreased by burn injury. This change was associated with a reduction in IGFBP-3 mRNA in liver but an increased expression of IGFBP-3 in kidney and muscle. Thermal injury also decreased the concentration of the acid-labile subunit (ALS) in plasma and ALS mRNA expression in liver. Finally, hepatic expression of IGFBP-related peptide-1 was increased twofold in liver but was unchanged in kidney or muscle of burn rats. These results characterize burn-induced changes in various components of the IGF system in select tissues and thereby provide potential mechanisms for alterations in the circulating IGF system and for changes in tissue metabolism.
insulin-like growth factor binding protein-1 and -3; mac25; acid-labile subunit; amino acids; rats
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THERMAL INJURY PRODUCES A NUMBER of well-characterized hormonal and metabolic alterations. One such hormonal alteration is the pronounced decrease in the circulating concentration of insulin-like growth factor (IGF)-I. A decrease in plasma IGF-I has been demonstrated by several investigators to occur early (within 24 h) after thermal injury and to remain reduced for several weeks thereafter (1, 16, 23, 32, 34). Furthermore, the burn-induced decrease in plasma IGF-I appears to be proportional to the severity of the insult (1, 32). The liver is the primary source of blood-borne IGF-I (24), and previous studies have demonstrated that hepatic synthesis and secretion of IGF-I are impaired in other catabolic-inflammatory conditions, such as sepsis and endotoxemia (8, 9, 22, 46). Hence, it is assumed, but not proven, that a similar defect in hepatic IGF-I synthesis occurs after thermal injury. Furthermore, a variety of extrahepatic tissues contain the peptide and mRNA for IGF-I (33, 46), suggesting that IGF-I functions as both a classical hormone and an autocrine-paracrine regulator. In this regard, tissue-specific changes in IGF-I peptide and mRNA have been previously reported in other catabolic conditions (8, 9, 38) and may impact upon metabolic pathways in those tissues.
IGF-I in the blood and various body fluid compartments is bound noncovalently to one of several IGF binding proteins (IGFBPs). The majority of the circulating IGF-I is bound to IGFBP-3 and the acid-labile subunit (ALS) to form a ternary complex (27, 37). Because of its large molecular weight, this complex is restricted to the vascular compartment and is believed to represent a storage reservoir for IGF-I (27, 37). In contrast, a relatively small amount of the total IGF-I in the blood is carried by IGFBP-1 (26). Because of the size of this binary complex, IGFBP-1 represents a potential mechanism for the translocation of IGF-I across the capillary endothelium (26). Although the exact functions of these binding proteins are not known, it is clear that they represent another mechanism by which the tissue availability of IGF-I can be modulated. In addition, several of the IGFBPs have IGF-independent effects (13, 21). To our knowledge, there are only three studies reporting IGFBP levels after thermal injury, and all have been performed in humans (16, 23, 34). All studies clearly demonstrate a decrease in the circulating concentration of IGFBP-3 at various times after burn. However, for IGFBP-1, two of the studies showed increased levels (23, 34), and the third reported no change (16). Burn-induced changes in tissue mRNA for these IGFBPs have not been reported.
The purpose of the present study was to characterize changes in IGF-I, IGFBP-3, and IGFBP-1 in the systemic circulation and in liver and selected extrahepatic tissues after acute (24-h) thermal injury in the rat. Burn is associated with alterations in the release of numerous hormones that are capable of modulating various components of the IGF system. The stimulation of hepatic IGF-I, IGFBP-3, and ALS synthesis is generally considered to be growth hormone (GH) dependent (7, 12, 35, 43), whereas insulinopenia has been reported to decrease IGF-I and IGFBP-3 and to increase IGFBP-1 levels (15, 23, 28). Elevated glucocorticoid levels also decrease IGF-I and increase IGFBP-1 (28, 29, 41). Finally, alterations in the concentration of dietary protein and amino acids can differentially regulate the hepatic synthesis of IGF-I and IGFBP-1 (20, 36). Therefore, we have also measured the plasma concentrations of these various hormones/substrates to begin to assess their potential role in regulating the IGF system in response to thermal injury.
![]() |
METHODS AND MATERIALS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animal preparation and experimental protocol.
Adult specific pathogen-free male Sprague-Dawley rats (285-310 g;
Charles River Breeding Laboratories, Cambridge, MA) were housed at a
constant temperature, exposed to a 12:12-h light-dark cycle, and
maintained on standard rodent chow and water ad libitum for 1 wk
before experiments were performed. All experiments were approved by the
Animal Care and Use Committee at the Pennsylvania State University
College of Medicine and adhered to the National Institutes of Health
guidelines for the use of experimental animals.
IGF-I determination. The concentration of total IGF-I in plasma was determined by a modified acid-ethanol (0.25 N HCl-87.5% ethanol) procedure with cryoprecipitation, and tissues were processed by use of acid homogenization and Sep-Pak (C-18) extraction (8, 9, 22, 46). Urine samples were concentrated tenfold by centrifugal concentration, and the dried sample was reconstituted with RIA buffer containing 0.25% BSA for IGF-I determination. IGF-I in plasma, urine, and tissues was determined by RIA. Recombinant human [Thr59]IGF-I was used for iodination and standards (Genetech, South San Francisco, CA). The ED50 for this assay is 0.03-0.08 ng/tube. The tissue protein concentration was determined by the biuret method, and tissue IGF-I content was expressed as nanograms of IGF-I per microgram of tissue protein.
The plasma concentration of free IGF-I was determined by centrifugal ultrafiltration (3). Briefly, the plasma samples were diluted 1:5 with Krebs-Ringer bicarbonate buffer (pH 7.4, with 5% BSA) and prefiltered through a 0.22-µm 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, Beverly, MA) and centrifuged at 300 g (1,500 rpm) at 37°C for 100 min. The ultrafiltrate was collected from 40 to 100 min of centrifugation and used for the IGF-I RIA.RNA extraction and Northern blotting. Total RNA was isolated with TRI reagent TR-118, as outlined by the manufacturer (Molecular Research Center, Cincinnati, OH). Twenty- to one hundred-microgram samples of total RNA were run under denaturing conditions in 1% agarose-6% formaldehyde gels. The running buffer was 1× HEPES. Northern blotting occurred via capillary transfer to Zeta-Probe GT blotting membranes (Bio-Rad Laboratories, Hercules, CA). An 800-bp probe from rat IGF-I (Peter Rotwein, St. Louis, MO), a 600-bp probe of the rat growth hormone receptor (GHR) comprising the extracellular domain, the putative transmembrane region, and a short section of the intracellular domain (Lawrence Mathews, Ann Arbor, MI), a 407-bp probe from IGFBP-1 and a 699-bp probe from IGFBP-3 (Shunichi Shimisaki, San Diego, CA), and a 1-kb probe from mouse mac25 (Mitsuo Kato, Kyoto, Japan) were labeled by use of a random primed DNA labeling kit (Roche Molecular Biochemicals, Indianapolis, IN). To probe for rat ALS, a 36-mer oligonucleotide was constructed. The complement of the second exon position 3843-3878 (5'-GAC GCT TCG GAG TGC GTT CCT GCT CAG ATC CAG CTC-3') was the sequence chosen for the oligonucleotide. A rat 18S oligonucleotide was used for normalization of RNA loading. Both oligonucleotides were synthesized by the Macromolecular Core Facility at the Pennsylvania State College of Medicine with a Perseptive Biosystems Expedite 8909 nucleic acid synthesizer. Each oligonucleotide was radioactively end-labeled with T4 polynucleotide kinase (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were prehybridized and hybridized at 42°C in 50% formamide-6× SSPE-5× Denhardt's-1% SDS-10% dextran sulfate-herring testis DNA (100 µg/ml), with the exception that ULTRAhyb (Ambion, Austin, TX) was used as the hybridization solution for rat ALS and mac25. All membranes were washed at room temperature twice in 2× SSC-0.1% SDS for 5 min and once in 0.1× standard sodium citrate (SSC)-0.1% SDS for 15 min. Additionally, membranes hybridized with rat IGF-I, GHR, or mac25 were washed at 55°C in 0.1× SSC-0.1% SDS for 15-30 min. Finally, membranes were exposed to a phosphoimager screen and the resultant data quantitated by ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Data for all probes were normalized to the level 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.
Ligand blotting.
IGFBP-3 in plasma was determined by Western ligand blot analysis (8, 9,
22, 46). Samples were subjected to SDS-PAGE without reduction of
disulfide bonds. The electrophoresed proteins were transferred onto
nitrocellulose in Tris-methanol-glycine buffer. Nitrocellulose sheets
were washed and then incubated overnight with radiolabeled IGF-I. The
nitrocellulose sheets were washed extensively in Tween 20, dried, and
autoradiographed with X-ray film (Kodak X-Omat AR, Eastman Kodak,
Rochester, NY) and intensifying screens (Du Pont, Wilmington, DE) at
70°C for 2-4 days.
Western immunoblotting. Plasma samples were separated on a 12.5% SDS-PAGE gel under nonreducing conditions, as previously described (46). 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 a 1:2,000 dilution of antiserum against rat IGFBP-1 at room temperature for 2 h. Antigen-antibody complexes were identified with goat anti-rabbit IgG tagged with horseradish peroxidase (Sigma, St. Louis, MO) and exposed to the enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL) for 1 min and to X-ray film for 10-30 s. Bands were scanned (Microtek ScanMaker IV) and quantitated with NIH Image 1.6 software. Representative samples from all experimental groups were electrophoresed on the same gel; data are expressed as a percentage of the control value.
IGFBP-3 proteolysis. Proteolysis was determined on plasma from control and burned rats (n = 5 for each group). Plasma samples were incubated either alone or mixed and incubated in 0.1 M Tris buffer (pH 7.4) at 37°C for 18 h. Samples were subsequently separated by SDS-PAGE, and IGFBPs were detected by ligand blotting, as described above. IGFBP-3 proteolysis was defined as a loss of IGFBP-3 by ligand blotting compared with control plasma. Plasma from a pediatric AIDS patient was used as a positive control for IGFBP-3 proteolysis.
Plasma glucose, hormone, and amino acid concentrations. The plasma glucose concentration was determined by means of a rapid analyzer (GL5, Analox Instruments, Lunenburg, MA). The plasma concentrations of rat insulin (Linco, St. Louis, MO), GH (Amersham), and corticosterone (Diagnostic Products, Los Angeles, CA) were determined by specific RIAs. An aliquot of plasma was deproteinized with sulfosalicylic acid, and the supernatant was used for amino acid analysis by ion-exchange HPLC (model 6300, Beckman Instruments, Fullerton, CA). Absorbance was measured at 440 and 570 nm after postcolumn ninhydrin treatment. S-(2-aminoethyl)-L-cysteine was used as an internal standard, and data acquisition and management were performed by Beckman System Gold 8.10.
Statistics. Data were obtained from two separate experimental series, each containing sham-control and burn rats (5-6 rats per group). Experimental values are presented as means ± SE. The numbers of rats per group are indicated in the figure legends. Data were analyzed with Student's t-test to determine treatment effect. Statistical significance was set at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasma IGF-I and IGFBPs, and IGF-I excretion.
The concentration of total IGF-I in plasma was decreased 38%
in rats ~24 h after thermal injury, compared with time-matched control animals (Fig. 1A). Burn
animals also showed a 65% decrease in the plasma concentration of free
IGF-I (Fig. 1B). As a result of these changes, the relative
amount of free IGF-I, compared with total IGF-I, decreased from 5.8 to
3.2% in burn rats (Fig. 1C).
|
|
Tissue IGF-I peptide and mRNA.
A concomitant reduction in IGF-I peptide content was also
detected in liver (43%) from burn rats (Fig.
3). Thermal injury decreased IGF-I in
muscles composed predominantly of mixed fast-twitch fibers. In this
regard, IGF-I content was decreased by 56% in the gastrocnemius and
69% in the extensor digitorum longus (EDL). The response in muscles
with a high proportion of slow-twitch fibers (heart and soleus) was
less pronounced. In burn rats, IGF-I content of heart was decreased
(28%), whereas there was no significant difference in IGF-I in the
soleus from sham-control and burn rats (Fig. 3). In contrast to the
above-mentioned changes, thermal injury increased the IGF-I content of
kidney by 36%.
|
|
Growth hormone receptor and GH binding protein mRNA. After hybridization, two bands were visualized: a 4.4-kb transcript encoding the GH receptor (GHR) and a 1.2-kb transcript encoding GH binding protein (GHBP) (data not shown). There was no significant difference between the expression of GHR mRNA in control and burned rats for either liver [control, 1.00 ± 0.6 vs. burn, 1.06 ± 0.18 arbitrary units (AU) of volume] or kidney (control, 1.00 ± 0.10 vs. burn, 1.24 ± 0.15 AU), as assessed by Northern blot analysis. There was a tendency for GHR mRNA in skeletal muscle to be decreased by thermal injury, but this change did not reach statistical significance (controls, 1.00 ± 0.09 vs. burn, 0.84 ± 0.05 AU). Likewise, there was no significant difference in GHBP mRNA expression between control and burned rats for liver, kidney, or skeletal muscle (data not shown).
Tissue IGFBPs and ALS.
IGFBP-1 mRNA expression was increased ~5-fold in liver and ~12-fold
in kidney (Fig. 5) in response to thermal
injury. IGFBP-1 mRNA expression could not be detected in skeletal
muscle by Northern blot analysis (data not shown).
|
|
|
Glucose, hormone, and amino acid concentrations. The plasma concentrations of glucose and several hormones capable of influencing the IGF system were determined. Thermal injury increased the concentration of both insulin (196 ± 30 vs. 422 ± 54 pmol/l; P < 0.05) and corticosterone (198 ± 16 vs. 326 ± 29 ng/ml; P < 0.05) compared with control values. A single-point determination of plasma GH was not different between groups (control, 18 ± 2 vs. burn, 19 ± 2 ng/ml). There was also no significant difference in the plasma glucose concentration between sham-control and burn rats (9.1 ± 0.4 vs. 9.5 ± 0.7 mM, respectively) at the 24-h time point.
The effect of burn injury on the concentration of individual plasma amino acids is presented in Table 1. Overall, burn rats demonstrated an 8% decrease in the concentration of total amino acids, but this change was not statistically significant. However, several individual amino acids did show significant decreases in response to thermal injury; these included glutamine (
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, we used a well defined thermal injury model in rats to characterize selected aspects of the IGF system in plasma and tissues. The plasma concentration of total IGF-I was markedly reduced in burn rats. This finding is consistent with a number of animal and human studies of burn injury (1, 16, 23, 32, 34). Moreover, we have extended these original observations by determining that burn decreased the absolute concentration of free IGF-I, as well as the relative amount of free IGF-I compared with the total IGF-I concentration in the plasma. The mechanism for the burn-induced decrease in circulating IGF-I has not been previously elucidated, but it could result from a decreased rate of synthesis and/or increased rate of removal from the blood. Our results confirm data from a previous report (19), which indicates that the content of hepatic IGF-I peptide is also reduced in burn rats. Burn also tended to decrease IGF-I mRNA expression in the liver, based on the combined abundance of the three major transcripts detected. However, whereas burn clearly decreased the smaller IGF-I transcripts (1.7 kb and 0.9-1.2 kb), the abundance of the 7.5-kb transcript was significantly increased. The overall decrease in hepatic total IGF-I mRNA was qualitatively similar to, albeit quantitatively smaller than, that observed in other catabolic conditions, including sepsis (22, 49), endotoxemia (29, 46), or cytokine-induced inflammation (8, 9). However, these latter insults produced a coordinated decrease in all detectable IGF-I transcripts. The reasons for this difference are unclear. It is also possible that an enhanced rate of IGF-I removal from the circulation contributes to the burn-induced decrease in plasma IGF-I. An enhanced rate of clearance may result from an increase in IGF-I binding by specific cells, such as leukocytes, enterocytes, and various renal cell types, which could be mediated by an upregulation of the IGF type I receptor and/or the amount of one or more of the IGF binding proteins. These possibilities were not examined in the present study; however, our data do indicate that the burn-induced decrease in plasma IGF-I is not a consequence of an increase in urinary IGF-I excretion.
GH is a potent stimulator of hepatic IGF-I synthesis and secretion. Therefore, burn-induced changes in plasma IGF-I may result because of a decrease in circulating levels of GH or an impaired responsiveness of tissues to GH. Although a single-point determination of plasma GH failed to detect any major difference between control and burn rats, we cannot exclude the possibility that pulsatile release of GH was impaired by thermal injury. Additionally, we were unable to detect significant alterations in GHR mRNA in any tissue examined. Therefore, it seems unlikely that the burn-induced changes in tissue IGF-I (or other GH-dependent processes described below) were mediated by the number of receptors for GH. Other studies have reported that the number of GHR in liver is either markedly decreased (5) or not altered (31) after the injection of endotoxin. The reason for the difference in GHR levels in these studies remains unclear but may be related to the severity of the insult and/or the time point at which samples were obtained. Regardless of the exact mechanism, both studies demonstrate the presence of endotoxin-induced hepatic GH resistance (5, 31), which has also been reported in response to thermal injury (19). Hence, the burn-induced decrease in plasma IGF-I and other GH-dependent processes appears most likely to result from a defect in the GH signaling pathway in liver. To our knowledge, there are no studies that examine the influence of catabolic stimuli on GH signal transduction in extrahepatic tissues.
IGF-I is an important anabolic agent in skeletal muscle, where it promotes protein synthesis and glucose uptake and inhibits protein degradation (2, 14). Burn rats showed a consistent decrease in the IGF-I peptide content in several fast-twitch skeletal muscles (gastrocnemius and EDL). There was no decrease in IGF-I peptide content in the slow-twitch soleus muscle, and there was a relatively small burn-induced decrease in heart. This fiber type selectivity is similar to that observed by other investigators for burn-induced changes in protein metabolism (10). In the gastrocnemius muscle, which has a fiber type similar to that of the whole body musculature, we also demonstrated a burn-induced decrease in IGF-I mRNA. The decrease in both IGF-I peptide and mRNA in skeletal muscle is similar to the response observed in other catabolic conditions (8, 9, 22, 46) and has been suggested to be at least partially responsible for the impairment in muscle protein balance and the negative nitrogen balance (22).
After thermal injury, there was an increase in IGF-I peptide but a decrease in IGF-I mRNA in kidney. This response suggests that burn injury increases the movement of IGF-I peptide from the circulation to the kidney rather than increasing renal IGF-I synthesis. These changes are similar to those reported in rats made acutely acidotic (4), but previous work by Horton et al. (18) indicates that rats are not acidotic or hypoxic at this time point after burn injury. However, these changes differ from those observed in other catabolic conditions that show an increase in both renal IGF-I peptide and mRNA levels (9, 29, 46). The burn-induced increase in renal IGF-I did not result in a concomitant increase in urinary IGF-I excretion. This suggests that IGF-I present in the kidney was largely sequestered or trapped in this tissue and that the reduction in plasma IGF-I did not result from an enhanced rate of excretion. Other catabolic conditions are characterized by an increase in IGFBP-1 content in the kidney (8, 9, 22, 46), and indeed, renal synthesis of IGFBP-1, as evidenced by an increase in IGFBP-1 mRNA, was increased in burn rats. Therefore, it seems likely that the renal sequestration of IGF-I occurred secondarily to the increase in IGFBP-1. We speculate that this burn-induced increase in renal IGF-I represents a beneficial response that may aid in maintaining renal perfusion and glomerular filtration (30).
The large majority of IGF-I in blood is carried bound to IGFBP-3 (37). Previous studies in humans report that thermal injury produces a rapid and sustained decrease in circulating IGFBP-3 (16, 23, 34). The current investigation demonstrates that a qualitatively similar IGFBP-3 response can be produced in rats. This decrease could result from an increased rate of clearance and/or a decreased rate of synthesis. In other catabolic conditions, some of the decrease in plasma IGFBP-3 may result from an enhanced rate of proteolysis (6). In the present study, however, we failed to detect a burn-induced increase in IGFBP-3 proteolysis. The decreased plasma concentration of IGFBP-3 in burn rats was associated with a comparable reduction in IGFBP-3 mRNA in liver. Previous studies indicate that exogenous administration of IGF-I increases hepatic IGFBP-3 mRNA in hypophysectomized rats (12); therefore, it is possible that the burn-induced decrease in hepatic IGFBP-3 mRNA results from the reduction in hepatic and/or circulating levels of IGF-I. Alternatively, we cannot exclude the possibility that a decrease in GH levels or the presence of hepatic GH resistance is responsible for the burn-induced decrease in hepatic IGFBP-3 mRNA. In contrast to liver, IGFBP-3 mRNA was increased in both kidney and muscle by burn injury. The threefold elevation in skeletal muscle IGFBP-3 mRNA expression was particularly dramatic. Based on the assumption there is a corresponding increase in IGFBP-3 protein in the muscle and surrounding interstitial fluid of burn rats and that elevations in IGFBP-3 appear to largely inhibit IGF-I mediated processes (37), it is possible that this local change is partially responsible for the metabolic disturbances seen in muscle. The mechanism for the increase in IGFBP-3 in muscle and kidney is not known.
A moderate decrease in the circulating concentration of ALS, the third
component of the ternary complex, was also observed in burn rats. This
decrease faithfully mimics the response seen in human subjects after
burn (23). In rats, however, the burn-induced decrease in hepatic ALS
mRNA was far more dramatic, with expression being reduced to <10% of
control levels. The greater reduction in hepatic ALS mRNA, compared
with plasma ALS protein, may be a consequence of its large molecular
weight and corresponding relatively long half-life in the blood. The
amount of ALS in the circulation is determined primarily by
GH-dependent transcriptional activation of the ALS gene within the
liver (35). The suppression of hepatic ALS mRNA in burn rats is
comparable to the decrease observed in hypophysectomized rats
completely lacking GH (12). Although our data suggest that such a
severe reduction in plasma GH is not present in burn rats, the same
response could be observed in animals with a defect in hepatic GH
signal transduction. Thermal injury increases the hepatic expression or
the systemic concentration of various proinflammatory cytokines (44).
Recently, Delhanty (7) has reported that relatively low doses of
interleukin-1 can inhibit GH-induced increases in ALS in primary
hepatocytes; hence, the overexpression of one or more cytokines remains
a possible mediator for the burn-induced decrease in ALS. Elevated
cytokine levels also inhibit IGF-I synthesis in isolated hepatocytes
(43), which is consistent with the reduction in abundance of the
smaller IGF-I mRNA transcripts observed in liver from burn rats.
An elevation in plasma IGFBP-1 is commonly observed in a variety of catabolic conditions (8, 9, 22, 46) and has been reported in some (23, 34), but not all (16), of the studies on burn trauma. In the current study, plasma IGFBP-1 was markedly and consistently elevated 24 h after thermal injury. We have extended these observations to reveal that IGFBP-1 mRNA expression was also elevated 5- to 10-fold in both liver and kidney. Known regulators for the elevations in IGFBP-1 include decreases in insulin (28), as well as increases in glucocorticoids and various cytokines (25, 28, 29). Because mild hyperinsulinemia was present in our burn rats, this hormone appears to be an unlikely candidate. Thus the latter group of agents remain as potential mediators for the elevation in blood and tissue IGFBP-1.
In addition to the six well characterized high-affinity IGFBPs, there are several new members of the IGFBP superfamily that have a relatively low sequence homology and IGF binding affinity. The impact of thermal injury on one of these peptides, IGFBP-rP1 (mac25 or IGFBP-7), was determined in the present study. We examined IGFBP-rP1 because of its reported ability to bind insulin and impair insulin-stimulated phosphorylation (45). It has been suggested that the overproduction of IGFBP-rP1 might play an important role in the pathogenesis of the insulin resistance that accompanies certain catabolic conditions (45). In this regard, thermal injury is known to produce a marked impairment of insulin action in muscle (39). Our data demonstrate that IGFBP-rP1 mRNA expression was elevated in liver, but not in muscle or kidney, in response to burn. Therefore, if IGFBP-rP1 is a mediator of the burn-induced muscle insulin resistance, it must be functioning via an elevation in the plasma concentration, as opposed to having an autocrine-paracrine effect.
The plasma concentrations of numerous individual amino acids were altered in response to burn. In general, the increased concentration of the branched-chain amino acids, as well as the elevation in tyrosine, phenylalanine, and 3-methylhistidine, strongly suggests a breakdown of myofibrillar protein (40). Similar changes have been reported previously in burn rats (11). However, the concentrations of other amino acids, such as glutamine, arginine, proline, glycine, and lysine, were decreased by burn. Hence, there was no significant change in the plasma concentration of total amino acids in burn rats. Therefore, it seems unlikely that burn-induced changes in total amino acid availability mediate the observed changes in the IGF system. Furthermore, IGFBP-1 secretion by HepG2 cells has been shown to be increased under conditions where the individual concentration of valine, leucine, isoleucine, phenylalanine, or histidine in the medium is decreased (20). However, the plasma concentration of each of these amino acids was increased in burn rats. Therefore, changes in individual amino acids do not appear to be important regulators of IGFBP-1 synthesis in this particular stress condition.
Based on the above-mentioned data, we speculate that the burn-induced decrease in plasma IGF-I occurs in the following manner. Hepatic expression of ALS is dramatically downregulated, resulting in a progressive decrease in plasma ALS. In addition, there is a concomitant impairment in hepatic synthesis and secretion of IGFBP-3, albeit less severe than that observed for ALS. The loss of ALS from the circulation prevents the formation of the ternary complex. Initially, there is still adequate IGFBP-3 available to bind IGF-I and form a binary complex. However, because of its smaller molecular weight, the IGF-I-IGFBP-3 complex is rapidly removed from the circulation, leading to a decline in plasma IGF-I. In addition, because of the upregulation of IGFBP-1, the relative percentage of IGF-I bound to this low-molecular-weight binding protein is expected to increase, thereby decreasing the circulating concentration of free IGF-I. Collectively, these changes would be expected to exacerbate the catabolism of muscle protein.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported in part by a grant from the National Institute of General Medical Sciences GM-38032.
![]() |
FOOTNOTES |
---|
We thank the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, for the generous gift of the IGF-I antibody, and Genentech (South San Francisco, CA) for the IGF-I used in these studies. In addition, we would like to express our sincere thanks to all the investigators who provided the cDNA probes used in this study: Drs. P. Rotwein (IGF-I), L. Mathews (GHR), S. Shimisaki (IGFBP-1 and -3) and M. Kato (mac25).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. H. Lang, Dept. of Cellular Molecular Physiology (H166), Pennsylvania State College of Medicine, Hershey, PA 17033-0859 (E-mail: clang{at}psu.edu).
Received 17 August 1999; accepted in final form 14 January 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abribat, T,
Brazeau P,
Davignon I,
and
Garrel DR.
Insulin-like growth factor-I blood levels in severely burned patients: effects of time post injury, age of patient and severity of burn.
Clin Endocrinol (Oxf)
39:
583-589,
1993[ISI][Medline].
2.
Bark, TH,
McNurlan MA,
Lang CH,
and
Garlick PJ.
Increased protein synthesis after acute IGF-I or insulin infusion is localized to muscle in mice.
Am J Physiol Endocrinol Metab
275:
E118-E123,
1998
3.
Bereket, A,
Wilson TA,
Blethen SL,
Fan J,
Frost RA,
Gelato MC,
and
Lang CH.
Effect of short-term fasting on free/dissociable IGF-I concentrations in normal human serum.
J Clin Endocrinol Metab
81:
4379-4384,
1996[Abstract].
4.
Bereket, A,
Wilson TA,
Kolasa AJ,
Fan J,
and
Lang CH.
Regulation of the insulin-like growth factor system by acute acidosis.
Endocrinology
137:
2238-2245,
1996[Abstract].
5.
Defalque, D,
Brandt N,
Ketelslegers JM,
and
Thissen JP.
GH insensitivity induced by endotoxin injection is associated with decreased liver GH receptors.
Am J Physiol Endocrinol Metab
276:
E565-E572,
1999
6.
Davenport, ML,
Isley WL,
Pucilowska JB,
Pemberton LB,
Lyman B,
Underwood LE,
and
Clemmons DR.
Insulin-like growth factor-binding protein-3 proteolysis is induced after elective surgery.
J Clin Endocrinol Metab
75:
590-595,
1992[Abstract].
7.
Delhanty, PJD
Interleukin-1 suppresses growth hormone-induced acid-labile subunit mRNA levels and secretion in primary hepatocytes.
Biochem Biophys Res Commun
243:
269-272,
1998[ISI][Medline].
8.
Fan, J,
Bagby GJ,
Gelato MC,
and
Lang CH.
Regulation of insulin-like growth factor I content and IGF-binding proteins by tumor necrosis factor.
Am J Physiol Regulatory Integrative Comp Physiol
269:
R1204-R1212,
1995
9.
Fan, J,
Wojnar MM,
Theodorakis M,
and
Lang CH.
Regulation of insulin-like growth factor (IGF)-I mRNA and peptide, and IGF binding proteins by interleukin-1.
Am J Physiol Regulatory Integrative Comp Physiol
270:
R621-R629,
1996
10.
Fang, CH,
James JH,
Ogle C,
Fischer JE,
and
Hasselgren PO.
Influence of burn injury on protein metabolism in different types of skeletal muscle and the role of glucocorticoids.
J Am Coll Surg
180:
33-42,
1995[ISI][Medline].
11.
Fang, CH,
Li BG,
Wang JJ,
Fischer JE,
and
Hasselgren PO.
Treatment of burned rats with insulin-like growth factor I inhibits the catabolic response in skeletal muscle.
Am J Physiol Regulatory Integrative Comp Physiol
275:
R1091-R1098,
1998
12.
Fielder, PJ,
Mortensen DL,
Mallet P,
Carlsson B,
Baxter RC,
and
Clark RG.
Differential long-term effects of insulin-like growth factor-I (IGF-I), growth hormone (GH), and IGF-I plus GH on body growth and IGF binding proteins in hypophysectomized rats.
Endocrinology
137:
1913-1920,
1996[Abstract].
13.
Frost, RA,
and
Lang CH.
Differential effects of insulin-like growth factor I (IGF-I) and IGF-binding protein-1 on protein metabolism in human skeletal muscle cells.
Endocrinology
140:
3962-3970,
1999
14.
Fryburg, DA.
Insulin-like growth factor I exerts growth hormone- and insulin-like actions on human muscle protein metabolism.
Am J Physiol Endocrinol Metab
267:
E331-E336,
1994
15.
Frystyk, J,
Hussain M,
Skjaerbaek C,
Schmitz O,
Christiansen JS,
Froesch FR,
and
Orskov H.
Serum free IGF-I during a hyperinsulinemic clamp following 3 days of administration of IGF-I vs. saline.
Am J Physiol Endocrinol Metab
273:
E507-E513,
1997
16.
Ghahary, A,
Fu S,
Shen YJ,
Shankowsky HA,
and
Tredget EE.
Differential effects of thermal injury on circulating insulin-like growth factor binding proteins in burn patients.
Mol Cell Biochem
135:
171-180,
1994[ISI][Medline].
17.
Herndon, DN,
Wilmore DW,
and
Mason AD.
Development and analysis of a small animal model simulating the human postburn hypermetabolic response.
J Surg Res
25:
394-403,
1978[ISI][Medline].
18.
Horton, JW,
White J,
Maass D,
and
Sanders B.
Arginine in burn injury improves cardiac performance and prevents bacterial translocation.
J Appl Physiol
84:
695-702,
1998
19.
Jeschke, MG,
Chrysopoulo MT,
Herndon DN,
and
Wolf SE.
Increased expression of insulin-like growth factor-I in serum and liver after recombinant human growth hormone administration in thermally injured rats.
J Surg Res
85:
171-177,
1999[ISI][Medline].
20.
Jousse, C,
Bruhat A,
Ferrara M,
and
Fafournoux P.
Physiological concentration of amino acids regulates insulin-like growth-factor-binding protein 1 expression.
Biochem J
334:
147-153,
1998[ISI][Medline].
21.
Lalou, C,
Lassarre C,
and
Binoux M.
A proteolytic fragment of insulin-like growth factor (IGF) binding protein-3 that fails to bind IGFs inhibits the mitogenic effects of IGF-I and insulin.
Endocrinology
137:
3206-3212,
1996[Abstract].
22.
Lang, CH,
Fan J,
Cooney R,
and
Vary TC.
Interleukin-1 receptor antagonist attenuates sepsis-induced alterations in the insulin-like growth factor system and protein synthesis.
Am J Physiol Endocrinol Metab
270:
E430-E437,
1996
23.
Lang, CH,
Fan J,
Frost RA,
Gelato MC,
Sakurai Y,
Herndon DN,
and
Wolfe RR.
Regulation of the insulin-like growth factor system by insulin in burn patients.
J Clin Endocrinol Metab
81:
2474-2480,
1996[Abstract].
24.
Lang, CH,
Frost RA,
Ejiofor J,
Lacy DB,
and
McGuinness OP.
Hepatic production and intestinal uptake of IGF-I: response to infection.
Am J Physiol Gastrointest Liver Physiol
275:
G1291-G1298,
1998
25.
Lang, CH,
Nystrom GJ,
and
Frost RA.
Regulation of insulin-like growth factor binding protein-1 in HepG2 cells by cytokines and reactive oxygen species.
Am J Physiol Gastrointest Liver Physiol
276:
G719-G727,
1999
26.
Lee, PDK,
Conover CA,
and
Powell DR.
Regulation and function of insulin-like growth factor-binding protein-1.
Proc Soc Exp Biol Med
204:
4-29,
1993[Abstract].
27.
Lewitt, MS,
Saunders H,
and
Baxter RC.
Bioavailability of insulin-like growth factors (IGFs) in rats determined by the molecular distribution of human IGF-binding protein-3.
Endocrinology
133:
1797-1802,
1993[Abstract].
28.
Lewitt, MS,
Saunders H,
and
Baxter RC.
Interaction of insulin, glucocorticoids, and protein kinase C in the regulation of insulin-like growth factor-binding protein-1 production by H4IIE hepatoma cells.
J Cell Physiol
166:
121-129,
1996[ISI][Medline].
29.
Li, YH,
Fan J,
and
Lang CH.
Role of glucocorticoids in mediating endotoxin-induced changes in insulin-like growth factor (IGF)-I and IGF binding protein-1.
Am J Physiol Regulatory Integrative Comp Physiol
273:
R1970-R1977,
1997.
30.
Lin, JJ,
Tonshoff B,
Bouriquet N,
Casellas D,
Kaskel FJ,
and
Moore LC.
Insulin-like growth factor-I restores microvascular autoregulation in experimental chronic renal failure.
Kidney Int
54, Suppl67:
S195-S198,
1998[ISI].
31.
Mao, Y,
Ling PR,
Fitzgibbons TP,
McCowen KC,
Frick GP,
Bistrian BR,
and
Smith RJ.
Endotoxin-induced inhibition of growth hormone receptor signaling in rat liver in vivo.
Endocrinology
140:
5505-5515,
1999
32.
Moller, S,
Jensen M,
Svensson P,
and
Skakkebaek NE.
Insulin-like growth factor 1 (IGF-1) in burn patients.
Burns
17:
279-281,
1991[ISI][Medline].
33.
Murphy, LJ,
Bell GI,
and
Friesen HG.
Tissue distribution of insulin-like growth factor I and II messenger ribonucleic acid in the adult rat.
Endocrinology
120:
1279-1282,
1987[Abstract].
34.
Nygren, J,
Sammann M,
Malm M,
Efendic S,
Hall K,
Brismar K,
and
Ljungqvist O.
Disturbed anabolic hormonal patterns in burned patients: the relation to glucagon.
Clin Endocrinol (Oxf)
43:
491-500,
1995[ISI][Medline].
35.
Ooi, GT,
Cohen FJ,
Tseng LYH,
Rechler MM,
and
Boisclair YR.
Growth hormone stimulates transcription of the gene encoding the acid-labile subunit (ALS) of the circulating insulin-like growth factor-binding protein complex and ALS promoter activity in rat liver.
Mol Endocrinol
11:
997-1007,
1997
36.
Pucilowska, JB,
Davenport ML,
Kabir I,
Clemmons DR,
Thissen JP,
Butler T,
and
Underwood LE.
The effect of dietary protein supplementation on insulin-like growth factors (IGFs) and IGF-binding proteins in children with shigellosis.
J Clin Endocrinol Metab
77:
1516-1521,
1993[Abstract].
37.
Rajaram, S,
Baylink DJ,
and
Mohan S.
Insulin-like growth factor-binding proteins in serum and other biological fluids: regulation and functions.
Endocr Rev
18:
801-83,
1997
38.
Rodriguez-Arnao, J,
Yarwood G,
Ferguson C,
Miell J,
Hinds CJ,
and
Ross RJM
Reduction in circulating IGF-I and hepatic IGF-I mRNA levels after caecal ligation and puncture are associated with differential regulation of hepatic IGF-binding protein-1, -2, and -3 mRNA levels.
J Endocrinol
151:
287-292,
1996[Abstract].
39.
Shangraw, RE,
Jahoor F,
Miyoshi H,
Neff WA,
Stuart CA,
Herndon DN,
and
Wolfe RR.
Differentiation between septic and postburn insulin resistance.
Metabolism
38:
983-989,
1989[ISI][Medline].
40.
Sjolin, J,
Stjernstrom H,
Friman G,
Larsson J,
and
Wahren J.
Total and net muscle protein breakdown in infection determined by amino acid effluxes.
Am J Physiol Endocrinol Metab
258:
E856-E863,
1990
41.
Skjaerbaek, C,
Frystyk J,
Grofte T,
Flyvbjerg A,
Lewitt MS,
Baxter RC,
and
Orskov H.
Serum free insulin-like growth factor-I is dose-dependently decreased by methylprednisolone and related to body weight changes in rats.
Growth Horm IGF Res
9:
74-80,
1999[ISI][Medline].
42.
Thissen, JP,
Triest S,
Moats-Staats BM,
Underwood LE,
Mauerhoff T,
Maiter D,
and
Ketelslegers JM.
Evidence that pretranslational and translational defects decrease insulin-like growth factor-I concentrations during dietary protein restriction.
Endocrinology
129:
429-435,
1991[Abstract].
43.
Thissen, JP,
and
Verniers J.
Inhibition by interleukin-1 and tumor necrosis factor-
of the insulin-like growth factor I messenger ribonucleic acid response to growth hormone in rat hepatocyte primary culture.
Endocrinology
138:
1078-1084,
1997
44.
Vindenes, HA,
Ulvestad E,
and
Bjerknes R.
Concentrations of cytokines in plasma of patients with large burns: their relation to time after injury, burn size, inflammatory variables, infection, and outcome.
Eur J Surg
164:
647-656,
1998[ISI][Medline].
45.
Yamanaka, Y,
Wilson EM,
Rosenfeld RG,
and
Oh Y.
Inhibition of insulin receptor activation by insulin-like growth factor binding proteins.
J Biol Chem
272:
30729-30734,
1997
46.
Wojnar, MM,
Fan J,
Li YH,
and
Lang CH.
Endotoxin-induced changes in IGF-I differ in rats provided enteral vs parenteral nutrition.
Am J Physiol Endocrinol Metab
276:
E455-E464,
1999