Greater potency of IGF-I than IGF-I/BP-3 complex in catabolic parenterally fed rats

K. R. Kritsch, D. J. Huss, and D. M. Ney

Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We compared the anabolic effects of recombinant human insulin-like growth factor I (rhIGF-I, 2.5 mg/kg) and equimolar amounts of rhIGF-I prebound to rhIGF binding protein-3 (rhIGF-I/BP-3) coinfused continuously with total parenteral nutrition (TPN) solution in dexamethasone (Dex, 70 µg/day ip)-treated male rats for 6 days. The four TPN groups included control, Dex, Dex + IGF-I, and Dex+IGF-I/BP-3. Pharmacokinetic analysis indicated reduced clearance of IGF-I when infused as IGF-I/BP-3 compared with free IGF-I (0.91 ± 0.09 vs. 2.01 ± 0.19 ml serum/min, P < 0.001) and this was associated with significantly greater serum IGF-I concentrations in the Dex+IGF-I/BP-3 group. Despite greater total serum IGF-I levels, infusion of free IGF-I produced greater anabolic responses than IGF-I/BP-3 based on body weight, nitrogen balance, and jejunal cellularity. Treatment with free IGF-I, but not IGF-I/BP-3, significantly reduced serum insulin and glucose levels that were elevated due to Dex. There were no significant differences in liver IGF-I mRNA levels between groups. Serum IGFBP-3 levels were elevated with infusion of IGF-I/BP-3 compared with IGF-I. These results indicate greater anabolic potency of IGF-I compared with IGF-I/BP-3 when administered by continuous parenteral infusion with TPN solution in catabolic rats.

total parenteral nutrition; dexamethasone; bioavailability; pharmacokinetics


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INSULIN-LIKE GROWTH FACTOR I (IGF-I) is an anabolic peptide that mediates the effects of growth hormone, shares structural homology with proinsulin, and promotes anabolism and other insulin-like actions. IGF-I action is modulated by at least seven IGF-binding proteins (IGFBPs) (5), with IGFBP-3 acting as the major reservoir of IGF-I in serum (4, 36). Currently there is much interest in investigating rhIGF-I as an anabolic agent in three clinical conditions: diabetes, growth disorders, and catabolic states associated with malnutrition (11). Although IGF-I has therapeutic potential to attenuate catabolism (10, 20), it has been shown to lose efficacy over time (25) and contribute to hypoglycemia and other side effects in humans (11, 21).

IGFBP-3 is the most abundant binding protein in adult serum (4). The ternary complex composed of IGF-I, IGFBP-3, and the acid labile subunit (ALS) prolongs the half-life of IGF-I in the circulation and modulates the availability of IGF-I to tissue receptors (34). In addition, IGFBP-3 has recently been shown to alter IGF-I action independent of sequestering IGF-I to prolong its half-life; however, in vitro research is controversial in demonstrating potentiation (13, 15) or inhibition of IGF-I action (28, 30, 39) by IGFBP-3. Moreover, clinical and experimental studies of chronic renal failure show an association between elevated levels of IGFBP-3 and impaired growth, suggesting that excess IGFBP-3 may inhibit IGF-I action (6). IGF-I and IGFBP-3 may have independent and conjunctive potential for growth.

Administration of IGF-I prebound to nonglycosylated IGFBP-3 (IGF-I/BP-3) is being studied as an alternative to administration of free IGF-I, as pharmacokinetic studies demonstrate that IGF-I bound to IGFBP-3 has a longer half-life (24) and is less likely to cause hypoglycemia compared with IGF-I (1, 2). Studies in which rats receive daily subcutaneous injections (2, 17) or a tail vein bolus of IGF-I or IGF-I/BP-3 (43) show that IGF-I/BP-3 is more effective than IGF-I in promoting bone formation (2), increasing lean body mass (2), preventing hypoglycemia (1), and enhancing wound healing (17). However, the relative effects of these growth factors have not been assessed when administered via continuous parenteral infusion.

We have evaluated the relative anabolic and tissue-specific potency of IGF-I/BP-3 and IGF-I in a rat total parenteral nutrition (TPN) model of dexamethasone (Dex)-induced catabolism. Dex, a synthetic glucocorticoid, was used to mimic the stressed condition seen in patients with elevated glucocorticoid levels. To gain insight into the clearance and potency of IGF-I/BP-3 and IGF-I, we first performed pharmacokinetic studies, followed by an experiment subjecting Dex-treated rats to continuous parenteral infusion of either growth factor. Our goal was to compare IGF-I/BP-3 and IGF-I in how each may affect whole body and tissue-selective anabolism as well as the endogenous IGF-I/IGFBP-3 system.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Animal facilities and protocols were approved by the University of Wisconsin-Madison Animal Care and Use Committee. Male Sprague-Dawley rats, ~ 240 g, were housed individually in stainless steel cages while acclimated to a 12:12-h light-dark cycle at 22°C with free access to a semipurified diet and water for 3 days. Animals were fasted 18 h before placement of jugular catheters on day 0. Infusion of TPN solution began on day 0. Body weight and grams of TPN solution infused were recorded daily. Because the rats were on TPN, feces were negligible in nitrogen excretion determination. Urine was collected at a final concentration of 0.01% boric acid, its volume was recorded, and it was stored at 4°C for nitrogen analysis. Nitrogen balance was calculated as the difference between nitrogen infused and nitrogen excreted. Nitrogen excretion was determined by microkjeldahl analysis as previously described (42).

Experimental Design

Experiment 1: bolus-clearance study. The purpose of this experiment was to test whether IGF-I prebound to BP-3 (rhIGF-I/BP-3), compared with rhIGF-I alone, prolongs the half-life of circulating IGF-I when administered with TPN solution in the parenterally fed rat. After placement of catheters, the animals were allowed to recover from surgery for 3 days while maintained with TPN. TPN infusion began gradually on day 0 (25 ml providing 27 kcal, 111 kcal/kg body weight). On day 1 through day 3 rats were given 55 g of TPN solution (52 ml, 56 kcal, 233 kcal/kg body weight) which provided adequate nutrition. On day 3, the rats received a bolus injection, via the jugular catheter, of either IGF-I or IGF-I/BP-3 in TPN solution (n = 5/treatment). The bolus delivered equimolar doses (100 µg) of IGF-I administered in 0.5 ml of TPN solution over 30 s. Time began after the growth factor was administered. The bolus injection was followed by TPN infusion without growth factors. The rats were anesthetized using metaphane, and tail vein blood draws were taken at baseline (2 min before the bolus) and then at 3, 5, 15, and 40 min. At 60 min the rats were killed by exsanguination. Serum was analyzed for IGF-I concentrations for pharmacokinetic analysis.

Pharmacokinetic parameters were determined for each rat by fitting the data to a bi-exponential equation that describes a two-compartment model, C = Ae-alpha t + Be-beta t (41). In this equation C is the concentration of IGF-I in serum and t is time. At t = 0, Co = A + B, where A and B represent constants corresponding to IGF-I concentrations in the distribution and elimination phase, respectively. The elimination phase describes a decline in plasma IGF-I concentrations due to the elimination of the drug; the distribution phase represents the rapid dispersion of IGF-I. Co represents the maximal serum concentration after the bolus. Area under the curve (AUC) was calculated using integration of the exponential function [AUC = (A/alpha )+(B/beta )] or the trapezoidal rule. The results indicate that either method yields the same significant differences in distribution constants (beta  and B), the elimination rate constant (alpha ), AUC, and clearance between treatment groups. We report group means ± SE as determined by integration of the exponential function because it more precisely represents AUC in the two-compartment model. Clearance (CL), the amount of serum that can be cleared of the drug/unit of time, was calculated as dose/AUC. Dose equals the amount of IGF-I administered in the bolus.

Experiment 2: coinfusion of IGF-I or IGF-I/BP-3 with TPN in Dex-treated rats. The purpose of this experiment was to compare the ability of rhIGF-I and rhIGF-I/BP-3 to induce whole body and tissue-selective anabolism in parenterally fed rats with Dex-induced catabolism. This model was chosen because it mimics a stress response, including elevated glucocorticoid levels, insulin resistance, and nitrogen wasting. These symptoms are often observed in clinical situations where TPN is used. The four TPN treatment groups included TPN control, Dex control, Dex+IGF-I, and Dex+IGF-I/BP-3. The final sample size was 4 to 6 rats per group. After catheterization, TPN infusion was gradually increased to provide complete nutrition by day 2 continuing through day 7 (see Fig. 1). Rats receiving growth factors were given equimolar amounts of IGF-I (2.5 mg/kg rhIGF-I or 12.5 mg/kg rhIGF-I/BP-3, courtesy of Celtrix Pharmaceuticals, Santa Clara, CA) coinfused continuously with TPN solution. This dose of IGF-I produces a submaximal growth response according to Tomas et al. (37). Dexamethasone-21-phosphate (Sigma, St. Louis, MO) was administered in saline as an intraperitoneal injection delivering 70 µg/day. Rats not given Dex received saline injections. Urine was collected from day 0 to day 7. On day 7, the rats were anesthetized by intramuscular injection delivering 80 mg ketamine and 8 mg xylazine/kg body weight and killed by exsanguination. Tissues, including jejunum, colon, liver, kidneys, spleen, and hindlimb muscle, were collected and immediately frozen in liquid nitrogen.


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Fig. 1.   Time line and experimental protocol to assess effects of coinfusion of insulin-like growth factor (IGF)-I or IGF-I/binding protein (BP)-3 with total parenteral nutrition (TPN) solution in dexamethasone (Dex)-treated rats. After central line catheterization on day 0, rats were maintained on TPN for 7 days while receiving daily ip injections of Dex. TPN controls received saline injections. IGF-I or IGF-I/BP-3 was coinfused with TPN solution beginning on day 1. TPN infusion was gradually increased from day 0 to day 2, after which adequate nutrition was provided at a constant rate delivering 55 g or 56 kcal/day. Animals were killed for tissue collection on day 7.

TPN solution preparation. A TPN solution was prepared aseptically using commercial preparations of amino acids plus electrolytes (8.5% Travasol, Baxter, Deerfield, IL), 60% dextrose (Baxter), 20% long-chain triglyceride lipid emulsion (Intralipid, Pharmacia, Clayton, NC), vitamins (Astra USA, Westborough, MA), and trace elements (Multitrace-4, American Regent Laboratories, Shirley, NY), as previously reported (23). The TPN solution contained (in g/l) 43 amino acids, 180 dextrose and 28 lipid, providing 32 and 68% of nonprotein energy from fat and carbohydrate, respectively. Animals received 55 g (52 ml) of TPN from day 2 to day 7, which provided 56 kcal/day and 359 mg nitrogen/day.

Serum hormone concentrations. Serum IGFs were removed from binding proteins by high-pressure liquid chromatography (HPLC) under acidic conditions. Total IGF-I concentrations were determined by a double antibody RIA (16). Materials included rhIGF-I as a standard, 125I-rhIGF-I (Amersham, Arlington Heights, IL), polyclonal antibody to human IGF-I (National Hormone and Pituitary Program, Baltimore, MD) as the primary antibody, goat anti-rabbit IgG, and normal rabbit serum (Antibodies, Davis, CA). Glucose concentrations in deproteinized blood were determined by the glucose oxidase technique (8). Serum insulin concentrations were determined using a commercially available RIA kit (Linco Research, St. Charles, MO).

Serum IGFBP concentrations. Serum IGFBPs were estimated by the Far Western ligand blotting procedure, a modified Western ligand blot (WLB) (18). In short, 2 µl of serum were subjected to SDS-PAGE (12.5% resolving gel, 4% stacking gel) and proteins were transferred to nitrocellulose by electroblotting. Incubation of the nitrocellulose with 3% Nonidet-P40, 1% BSA, and 0.1% Tween 20 was necessary for quenching before hybridization with 125I-IGF-I (Amersham) overnight. The nitrocellulose was washed in 0.1% Tween 20 and saline buffer and exposed to film. Bands present in the 38- to 43-kDa range, showing as a triplet, correspond to variations in glycosylation of IGFBP-3 (19). The bands were quantitated by densitometry (PDI, Huntington Station, NY).

IGFBP-3 in the complex is nonglycosylated and appears at a smaller molecular weight than 38 to 43 kDa. To characterize the size range of IGFBP-3 from the complex, we performed an immunoblot using a goat polyclonal IgG specific for IGFBP-3 and non-cross-reactive with IGFBP-1, -2, -4, -5, -6 and -7 (Santa Cruz Biotechnology, Santa Cruz, CA) and a WLB using the purified complex and serum from a rat treated with IGF-I/BP-3. These results indicate that the purified IGFBP-3 in the complex, as well as in the serum of rats treated with IGF-I/BP-3, appears in the 30- to 34-kDa range as previously described by Sommer et al. (34).

IGF-I mRNA protection assay. An exon-2-derived cDNA probe was used to generate an antisense RNA probe to detect liver IGF-I mRNA by protection assay (RPA). The probe consisted of a 464 bp-fragment containing 238 bp of exon 3 and 4 and 226 bp of exon 2 sequence subcloned into pGEM-4Z, previously described by and courtesy of M. L. Adamo (J. Shermer et al., 33). The plasmid was linearized with EcoR I, and T7 RNA polymerase was used for transcription of the antisense RNA probe. Ribosomal 18S mRNA antisense template (Ambion, Austin, TX) was transcribed and used as a control. Ten micrograms of liver total RNA were used in the RPA (Hybspeed RPA Kit, Ambion) and run on a 5% acrylamide/8 M urea denaturing gel. RNA integrity was confirmed by ethidium bromide staining of 28S and 18S ribosomal RNAs on an agarose/formaldehyde gel. The exon 2 probe yielded a doublet at 305 nt, a band at 290 and 238 nt (33). These liver RNA samples had an extraneous band at ~ 270 nt, an artifact seen only in intravenously fed rats. The 18S probe appeared as a single band at 80 nt. The two probes were not used in the same hybridization reaction, because doing so would have resulted in a variety of unexplainable bands. Gels were done in duplicate or triplicate, repeating the same liver RNA samples with 3-4 samples per treatment. Bands were quantitated using phosphor imaging (Packard Instrument, Meridan, CT). Degrees of difference in band intensity were calculated with the TPN control set to one. Statistical analysis assessed the degrees of difference averaged among the gels.

Jejunum analysis. We compared the effects of IGF-I and IGF-I/BP-3 on TPN-induced jejunal atrophy in the catabolic state. The jejunum, anatomically defined as extending from the ligament of Treitz to 25 cm proximal to the cecum, was excised from each rat. Total jejunum length and weight were determined after intestinal contents were removed with a saline rinse. The first 10 cm distal to the ligament of Treitz were used for the determination of mucosal wet and dry weight; the next 10 cm were immediately frozen in liquid nitrogen and later made into mucosal homogenates that were assayed for protein [bicinchoninic acid (BCA) colorimetric assay, Pierce Chemical, Rockford, IL] and DNA (22) content. A 1-cm segment of jejunum was rinsed, fixed in 10% buffered Formalin, embedded in paraffin, and stained with hematoxylin and eosin. A light microscope was used for morphometric measurement of muscularis depth, crypt depth, villus height, and density (29).

Statistical Analysis

The data were analyzed by a one-way ANOVA using generalized linear models in SAS (version 6.12, SAS Institute, Cary, NC, 1996). When F tests from ANOVA indicated significant differences (P < 0.05), a multiple comparison was performed to compare group means using the least significant difference technique. Changes in body weight were assessed by repeated-measures analysis. The data are presented as means ± SE. Means with unlike superscripts indicate a significant difference (P < 0.05).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pharmacokinetic Parameters

Pharmacokinetic parameters are presented in Table 1 and Fig. 2. The maximum concentration of IGF-I in serum after the bolus injection (Co) was not significantly different between treatments, confirming equality in dosing. Recombinant human IGF-I administered in free form was cleared more rapidly from serum compared with IGF-I when administered prebound to BP-3, 2.01 ± 0.19 and 0.91 ± 0.09 ml serum/min, respectively, P < 0.05. AUC was significantly greater in rats given a bolus of IGF-I/BP-3 vs. IGF-I. The reduced clearance of IGF-I/BP-3 compared with IGF-I was mainly associated with variance in IGF-I dispersion during the distribution phase (-alpha ). The rates of elimination (-beta ) were significantly different, but the magnitude of the difference was reduced compared with the distribution phase. These data suggest that, when IGF-I/BP-3 is administered with TPN solution, serum IGF-I concentrations are elevated due to reduced clearance compared with when an equimolar bolus of free IGF-I is given.

                              
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Table 1.   Pharmacokinetic parameters from rats receiving an iv bolus of IGF-I or IGF-I/BP-3 delivering 100 µg of IGF-I



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Fig. 2.   Intravenous bolus-clearance of IGF-I and IGF-I/BP-3. Catheterized rats received an iv bolus dose of IGF-I or IGF-I/BP-3 containing 100 µg of IGF-I. Tail vein blood draws were taken at 2 min before the bolus injection and then at 3, 5, 15, 40, and 60 min post injection. The semilog plot presents mean serum IGF-I levels ± SE (n = 5/group). Serum IGF-I concentrations were significantly increased after iv injection of IGF-I/BP-3 compared with IGF-I. Means with different letters are significantly different (P < 0.05).

Serum Hormones

The concentrations of total IGF-I, glucose, and insulin in serum are shown in Fig. 3. Exogenous growth factor treatment significantly increased serum IGF-I concentrations compared with TPN controls. Consistent with the results from the bolus-clearance study, the concentration of IGF-I in serum after 6 days of growth factor treatment was significantly greater with IGF-I/BP-3 compared with free IGF-I.


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Fig. 3.   Serum total IGF-I, insulin, and glucose concentrations in Dex-treated, parenterally fed rats receiving IGF-I or IGF-I/BP-3 coinfused continuously with TPN solution for 6 days. TPN and Dex controls are included. Values are means ± SE (n = 4-6/group). Means with different letters are significantly different (P < 0.05).

Dex treatment without IGF-I increased serum insulin concentrations approximately fivefold and serum glucose concentrations ~35%. Treatment with free IGF-I, but not IGF-I/BP-3, counteracted Dex-induced hyperinsulinemia and hyperglycemia such that animals given Dex+IGF-I showed reduced serum insulin and glucose concentrations that were not significantly different from TPN controls.

Serum IGFBPs. Figure 4A shows a WLB with three serum samples from each group; this gel is representative of all samples analyzed. The histogram, Fig. 4B, reflects all densitometric units relative to the TPN control group. The IGF-I/BP-3 complex utilizes a nonglycosylated form of BP-3, which migrates in the 30- to 34-kDa region compared with endogenous glycosylated IGFBP-3, which migrates at 38 to 43 kDa (34). The infusion of IGF-I/BP-3 resulted in an intense band in the 30- to 34-kDa region. The sum of band intensity in the 30- to 34-kDa (exogenous) and the 38- to 43-kDa (endogenous) regions suggests that infusion of IGF-I/BP-3 resulted in IGFBP-3 levels ~1.5 times those of rats receiving IGF-I. IGF-I infusion stimulated endogenous IGFBP-3 production despite Dex-induced suppression of BP-3 compared with TPN controls. IGF-I/BP-3 significantly reduced endogenous serum IGFBP-3. There were no significant differences in densitometry readings in the 24- to 34-kDa region among the IGF-I, TPN, and Dex control groups. Serum concentrations of IGF-I were significantly correlated with the 38-kDa region (r = 0.777, P < 0.0001, n = 15).


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Fig. 4.   A Western ligand blot (WLB) of serum IGFBP levels in Dex-treated, parenterally fed rats receiving IGF-I or IGF-I/BP-3 coinfused continuously with TPN solution for 6 days. TPN and Dex controls are included. A: two microliters of rat serum were subjected to SDS-PAGE, and proteins were transferred to nitrocellulose and probed with 125I-IGF-I. The triplet in the 38- to 43-kDa region corresponds to glycosylated BP-3s. Bands in the 30- to 34-kDa region in rats treated with IGF-I/BP-3 correspond primarily to nonglycosylated IGFBP-3. This gel is representative of all samples analyzed. B: a histogram showing quantitation of the WLB autoradiographic data pertaining to all rat serum samples in the 38- to 43-kDa region. Data are projected relative to the TPN control. Values are adjusted means ± SE. Means with different letters are significantly different (P < 0.05).

Liver IGF-I mRNA RPA. To determine whether changes in circulating levels of IGF-I or BP-3 due to infusion of IGF-I or IGF-I/BP-3 were accompanied by changes in hepatic IGF-I mRNA levels, an RPA for IGF-I mRNA was conducted. There were no significant differences in liver IGF-I mRNA expression across all groups (Fig. 5A). A trend existed, suggesting that Dex treatment alone increased liver IGF-I mRNA compared with TPN controls (Fig. 5B). Growth factors administered concurrently with TPN did not significantly alter IGF-I mRNAs. There were no significant differences in the ribosomal 18S mRNA control, suggesting equal RNA loading across all lanes (Fig. 5C).


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Fig. 5.   RNA protection assays utilizing an IGF-I exon-2 derived antisense RNA probe (A) and a ribosomal 18S antisense control probe (C). Lanes labeled M represent various sizes of 32P-labeled RNA fragments. A: ten micrograms of total liver RNA from all parenterally fed rats were hybridized with 20,000 cpm of the 32P-labeled rat exon-2 IGF-I mRNA probe, followed by RNase digestion and electrophoresis of protected hybrids. The lane labeled + or - represents 10 µg of yeast RNA hybridized with the probe and treated with or without RNase, respectively. The gel was subjected to phosphor image analysis. B: the gels were run in duplicate or triplicate. The digital light units (DLUs) gathered by phosphor image analysis were computed as a degree of difference of the average units for the three TPN control samples. The values, representing IGF-I mRNA abundance, are means ± SE of the degree of difference. There were no significant differences among the groups (n = 3-4/group). C: ten micrograms of total liver RNA from the same animals as in the exon-2 IGF-I mRNA RPA. Samples were hybridized with 1,000 cpm of the antisense ribosomal 18S mRNA control probe. Similar controls and markers were used as described. There were no significant differences in DLUs among the groups, suggesting equal loading of RNA to all wells.

Body weights and nitrogen balance. There were no significant differences in body weights due to growth factor and/or Dex treatment until day 2. On day 7, the final body weights were as follows: TPN control > Dex+IGF-I > Dex + IGF-I/BP-3 > Dex control. The TPN controls and Dex+IGF-I final body weights (253 ± 7 g and 242 ± 4 g) were significantly greater than the Dex controls (221 ± 5 g) but were not significantly different from each other. The Dex+IGF-I/BP-3 group body weights (234 ± 2 g) were not significantly different from the Dex or Dex+IGF-I group. Repeated-measures analysis reports a time-by-treatment effect (P < 0.0001) suggesting that over time, the body weight patterns for the groups were significantly different. Body weight changes analyzed over 6 days (day 1 through day 7) show that the TPN controls and the Dex+IGF-I rats gained an average of 5 g (5 ± 5 and 5 ± 2, respectively), whereas the Dex+IGF-I/BP-3 and the Dex group showed a significant loss of body weight, 6 ± 2 and 18 ± 3 g, respectively (Fig. 6A). Treatment with IGF-I/BP-3 significantly attenuated the weight loss induced by Dex, although the magnitude of the anabolic effect was significantly less compared with treatment with free IGF-I.


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Fig. 6.   Net changes in body weight (A) and cumulative nitrogen balance (B) over 6 days (day 1 through day 7) in Dex-treated, parenterally fed rats receiving IGF-I or IGF-I/BP-3 coinfused continuously with TPN solution. TPN and Dex controls are included. Nitrogen balance was strongly correlated with body weight changes (r = 0.798, P < 0.0001). Values are means ± SE (n = 4-6/group). Means with different letters are significantly different (P < 0.05).

There were no significant differences in cumulative TPN and nitrogen intake among the groups (P > 0.10). Animals given free IGF-I had decreased nitrogen excretion (2,092 ± 75 mg N) and increased cumulative nitrogen retention (158 ± 68 mg N), which was associated with a greater body weight gain (5 ± 2) compared with rats given IGF-I/BP-3 (N exc: 2,631 ± 88 mg N; cumulative N retention: -232 ± 101 mg N; body weight change: -6 ± 2 g), (Fig. 6B). Body weight change over 6 days was strongly correlated with cumulative nitrogen retention (r = 0.798, P < 0.0001, n = 19). These data suggest a greater anabolic potency of free IGF-I than IGF-I/BP-3 when continuously infused.

Organ and tissue determinations. Relative tissue weights for spleen, kidney, liver and colon showed significant treatment effects and are shown in Table 2. Dex treatment decreased relative spleen weight compared with TPN controls; growth factor treatment was not differentiated from Dex treatment. Relative kidney weights were increased due to Dex treatment compared with TPN controls; growth factor treatment was not differentiated from Dex treatment. Dex treatment increased relative liver weight compared with TPN controls. IGF-I, but not IGF-I/BP-3, significantly attenuated the increase in liver mass due to Dex. Differences in absolute hindlimb muscle weight did not reach significance at P < 0.05; however, the data suggest that both IGF-I and IGF-I/BP-3 attenuate Dex-induced muscle catabolism (IGF-I: 1.84 ± 0.08 g, IGF-I/BP-3: 1.77 ± 0.09 g, Dex: 1.56 ± 0.06 g, TPN control: 1.85 ± 0.05, P = 0.0619). There were no significant differences between treatments for relative hindlimb muscle weights, suggesting that Dex-induced loss of muscle mass or IGF-I-induced gain was proportional to changes in body weight. Relative colon weights were decreased in the rats receiving Dex (average 0.40 g/100 g body weight) compared with the TPN controls (0.54 ± 0.04 g/100 g body weight).

                              
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Table 2.   Relative organ and tissue weights in Dex-treated, parenterally fed rats receiving IGF-I or IGF-I/BP-3 coinfused continuously with TPN solution for 6 days

Jejunum. A 10-cm segment of jejunum was analyzed for mass, cellularity, and histology (Table 3). Animals treated with free IGF-I had significantly greater intact jejunal mass (22-36% increase) compared with TPN controls. This trend was also exhibited in the wet and dry weights of jejunal mucosa, where IGF-I increased wet and dry weights > 40% compared with the other groups. Greater increases in mucosal DNA relative to protein content suggest that the increase in jejunal mass due to treatment with free IGF-I was associated with greater mucosal cellularity as well as with cellular hypertrophy. Dex+IGF-I rats had significantly greater villus height compared with the other groups; there were no significant differences between groups in either crypt depth or villus density. In summary, jejunal mass, villus height, and mucosal cellularity were significantly greater with IGF-I compared with IGF-I/BP-3, suggesting greater potency of IGF-I to attenuate the intestinal atrophy induced by TPN.

                              
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Table 3.   Jejunal mass, cellularity, and histology in Dex-treated, parenterally fed rats receiving IGF-I or IGF-I/BP-3 coinfused continuously with TPN solution for 6 days


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IGFBP-3 is the primary carrier of IGF-I in serum and is an important regulator and modulator of IGF-I action. IGFBP-3 action is paradoxical in that it potentiates IGF-I action by increasing IGF-I half-life; however, IGFBP-3 also inhibits IGF-I action by decreasing the availability of IGF-I to its receptor and by IGF-independent actions (19). This study supports and extends previous work (1, 43) showing that the clearance of IGF-I is reduced when infused as IGF-I/BP-3 compared with free IGF-I. Although serum IGF-I concentrations were significantly greater in Dex-treated rats receiving IGF-I/BP-3 compared with free IGF-I when coinfused continuously with TPN solution, anabolic response was reduced. This study suggests that IGF-I/BP-3 is not as efficacious as IGF-I in a whole animal TPN model of continuous infusion. This conclusion is drawn from three lines of evidence: whole body, metabolic, and tissue-specific parameters of growth.

Administration of either IGF-I or IGF-I/BP-3 significantly attenuated Dex-induced growth retardation; however, the potency for attenuating Dex-induced catabolism was significantly greater for free IGF-I compared with IGF-I/BP-3. Whole body growth was highly correlated with cumulative nitrogen balance among the groups, suggesting that differences in body weight indicate changes in lean body mass (Fig. 6). We conclude that either IGF-I or IGF-I/BP-3 preserves lean body mass in proportion to body weight. These data are consistent with evidence that administration of IGF-I/BP-3 (1, 2) or IGF-I (42) promotes increased lean body mass.

Metabolic parameters show that rats receiving Dex had increased serum insulin and glucose concentrations compared with TPN controls, suggesting a decrease in insulin sensitivity (Fig. 3). These results are consistent with observations made by Rizza et al. (31) and have been reported previously by our lab (26). IGF-I reduced serum insulin and glucose concentrations to TPN control levels, but IGF-I/BP-3 did not. These data suggest that free IGF-I, but not IGF-I/BP-3, reversed Dex-induced insulin resistance.

In the gastrointestinal tract, IGF-I was clearly more potent than IGF-I/BP-3 in maintaining gut structure (Table 3). IGF-I reversed TPN-induced decreases in jejunal mass, mucosal dry weight, protein and DNA, and villus height. Jejunal parameters were not significantly different in the rats treated with IGF-I/BP-3 compared with the TPN and Dex controls. These data suggest that IGF-I, when continuously infused complexed to BP-3, is unable to promote IGF-I action in the jejunum as did free IGF-I. The jejunal stimulation observed with IGF-I infusion is similar to other studies suggesting that the gut is sensitive to free IGF-I (29, 40, 44). It is possible that continuous infusion of IGF-I/BP-3, a molecule too large to cross the vasculature (7), does not release IGF-I from the complex in sufficient amounts to promote jejunal growth.

IGF-I, but not IGF-I/BP-3, attenuated the Dex-induced increase in relative liver weight. Dex decreased relative colon and spleen weights and increased relative kidney weight, but neither growth factor significantly attenuated these changes. These data suggest that the liver and jejunum exhibit increased sensitivity to free IGF-I. The net biological response is the outcome of a balance between IGF-I interactions with IGFBPs and IGF-I receptors. Clearly, the IGFBPs play an important role in the modulation of IGF-I action to target tissues.

These data demonstrate that there are advantages in potency and tissue specificity when free IGF-I, compared with IGF-I/BP-3, is administered by continuous parenteral infusion. The elevated serum total IGF-I concentrations associated with IGF-I/BP-3 infusion suggest that, if systemic IGF-I circulates with a decreased clearance rate, we ought to detect potential advantages with the IGF-I/BP-3 complex detected by others (1, 2, 43). So why is continuous parenteral administration of IGF-I associated with a greater magnitude of growth than IGF-I/BP-3? One major determinant of the relative efficacy of IGF-I and IGF-I/BP-3 is the accessibility of free IGF-I to the IGF-I receptor. Research has shown that IGFBP-3 has a greater affinity for IGF-I than the type I IGF-I receptor and that a 30-kDa protease specifically cleaves IGFBP-3, resulting in a decreased affinity of IGF-I for BP-3 and an increased affinity for the receptor (19). A potential explanation for decreased growth with infusion of IGF-I/BP-3 is that the IGF-I/BP-3 complex may undergo proteolysis at a slower rate compared with the endogenous ternary complex. This would result in IGF-I remaining unavailable to receptors, while being reserved in circulation. This is consistent with increased total serum IGF-I, elevated IGFBP-3 levels, and lack of jejunal growth with continuous infusion of IGF-I/BP-3.

Alternatively, the environment in which the growth factors were administered may explain why the IGF-I/BP-3 complex was not as effective as IGF-I in promoting anabolism in the current study. The evidence suggests that the affinity of IGF-I to the nonglycosylated synthetic IGFBP-3 or glycosylated endogenous IGFBP-3 is not significantly different (35). It is possible that mixing IGF-I/BP-3 with the TPN solution containing an admixture of protein, carbohydrate, fat, vitamins, and minerals may alter the affinity by which IGF-I binds to BP-3. If mixing with TPN solution increased the affinity of IGF-I to IGFBP-3, this may contribute to the decreased IGF-I bioavailability to receptors and the reduced anabolism in this model.

Another explanation for the reduced anabolic potency of IGF-I/BP-3 involves the IGF-I-independent actions of IGFBP-3. In vitro studies suggest that IGFBP-3, whether glycosylated or nonglycosylated (12), truncated or not (32), may potentiate or inhibit IGF-I action by binding to cell surface-associated proteins. IGFBP-3 has been shown to induce apoptosis in a dose-dependent manner in a prostate cancer cell line through an IGF to IGF-I receptor-independent pathway (30). Similarly, in fibroblast cells, high concentrations of IGFBP-3 dissociated IGF-I from its receptor, resulting in limited IGF-I action (28). Overexpression of IGFBP-3 in IGF-I receptor-negative mouse fibroblasts inhibited cell growth (39). On the other hand, studies suggest that the binding of IGFBP-3 to cell surface-associated proteins may facilitate the availability of IGF-I to the IGF-I receptor, thus potentiating IGF-I action (14, 15). On reviewing such studies, Jones concluded that higher concentrations of IGFBP-3 may inhibit IGF-I action; therefore, an optimal ratio of IGFBP-3 to IGF-I is required to produce a potentiating effect (19).

In the current study, IGF-I/BP-3 was administered in a 1:1 ratio, but over time, circulating levels of IGFBP-3 increased as indicated by WLB of serum samples taken after six days of growth factor administration (Fig. 4). Further analysis of serum samples collected at 3 and 5 min after the intravenous bolus injection of IGF-I/BP-3 showed BP-3 levels twofold above those observed in the rats receiving an IGF-I bolus (data not shown). This suggests an accumulation of BP-3 in circulation during the continuous parenteral infusion of IGF-I/BP-3. We conclude that the accumulation of such high levels of IGFBP-3 may inhibit IGF-I action by reducing IGF-I bioavailability to its receptor or by IGF-I-independent actions, as noted in vitro (39).

Consistent with the notion that IGF-I regulates expression of its primary carrier protein IGFBP-3, circulating levels of IGF-I and IGFBP-3 were positively correlated in the current study. To gain insight into potential coordinate regulation of hepatic IGF-I production and circulating levels of IGF-I and IGFBP-3 in rats infused with IGF-I, we assessed liver IGF-I mRNA expression. The administration of either IGF-I or IGF-I/BP-3 did not downregulate liver IGF-I mRNA, which reflects endogenous circulating IGF-I in the current study where nutrition was well controlled. Liver IGF-I mRNA levels were not correlated with serum levels of IGF-I or IGFBP-3, or with changes in body weight as reported in orally (27) and parenterally fed rats (42). The regulation of endogenous IGF-I and IGFBP-3 production is likely very complex in the current model system and warrants further investigation.

To conclude, this study finds no advantage to continuous parenteral infusion of IGF-I/BP-3 compared with free IGF-I in rats with Dex-induced catabolism that are maintained with TPN. Continuous coinfusion of IGF-I with TPN solution attenuates Dex-induced catabolism to a greater degree than does IGF-I/BP-3 in the rat. In support of our results and the notion that the availability of free IGF-I to tissue receptors is a major determinant of anabolic response, IGF-I analogs that have a decreased binding affinity for the IGFBPs and the IGF-I receptor are more potent than IGF-I when given by injection or by continuous subcutaneous infusion via osmotic pumps (3, 38). Our results contrast with studies comparing free IGF-I to IGF-I/BP-3 when infused subcutaneously. This difference may be attributed to our method of growth factor administration, i.e., continuous parenteral infusion with a nutrient admixture. Moreover, recent evidence suggests that a continuous subcutaneous infusion of IGF-I/BP-3 is an effective means of improving insulin sensitivity in humans with type I diabetes (9). We conclude that the anabolic potency of IGF-I depends largely on the bioavailability of IGF-I to its receptors and tissue specificity to IGF-I.


    ACKNOWLEDGEMENTS

We thank Mike Grahn, Melanee Clark, Elizabeth Dahly, and Melanie Gillingham for their expert technical assistance and animal care, and Judy Kozminski for graphic artistry. We thank Dr. Ronald Burnette for expert advice in pharmacokinetics and Dr. Martin Adamo for assistance with the RPA.


    FOOTNOTES

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-42835 and T32-DK-07665 and by funds from the College of Agricultural and Life Sciences, University of Wisconsin-Madison.

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: D. M. Ney, Department of Nutritional Sciences, University of Wisconsin-Madison, 1415 Linden Drive, Madison, WI 53706 (E-mail: ney{at}nutrisci.wisc.edu).

Received 4 May 1999; accepted in final form 27 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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