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The deleterious effects of corticosteroids on growth have been known for a long time(1) and are a continuing cause for concern in pediatric patients. High plasma levels of corticosteroids increase leucine turnover and oxidation, indicating whole body protein catabolism, in human adult volunteers(2, 3) and in infants with bronchopulmonary dysplasia(4). Plasma amino acid levels are increased in infants under corticosteroid treatment(5). This is in part due to increased breakdown of muscle protein(6). Recently, there has been increasing interest in the use of recombinant polypeptide growth factors (GH, IGF-I, and IGF-I analogs/derivatives) to reverse various states of catabolism, among which is corticosteroid-induced protein catabolism. As early as 1952, Selye(7) demonstrated improved growth in glucocorticoid-treated rats if bovine GH was administered. A reversal of protein catabolism by increased protein synthesis has been proposed as mechanism for the anabolic actions of GH in corticosteroid-treated adults(8). Administration of IGF-I improves nitrogen balance in fasted lambs(9), and decreases whole-body leucine turnover in healthy human volunteers, suggesting decreased protein breakdown(10). We used DEX-treated rapidly growing piglets as an animal model for corticosteroid-induced protein catabolism in pediatric patients. Piglets were chosen for this model, because DEX treatment produced changes in leucine kinetics in piglets (our own data) similar to those reported in humans(2–4), and because the porcine GH/IGF-axis is similar to that in man-the porcine IGF-peptide is identical to human IGF-I(11). IGF-I was chosen as a possible antagonist for corticosteroid-induced protein catabolism in our study because it mediates GH action(12), interacts readily with the insulin receptor to lower glucose levels(13), and could thus be expected to attenuate corticosteroid-induced glucose intolerance. Our goal was to determine whether IGF-I reverses corticosteroid-induced protein catabolism in growing piglets, and whether IGF-I attenuates corticosteroid-induced hyperglycemia.

METHODS

Study design. We did three experiments on each piglet, at weekly intervals: baseline, DEX treatment, and DEX and IGF-I treatment combined, the latter two studies being carried out in a crossover study design. On study days, the piglets were sedated by intramuscular administration of 40 mg of azaperone (Stresnil®, Janssen Pharmaceutica BV, Tilburg, The Netherlands) and 5 mg of diazepam (t = 0 min). Azaperone is a butyrophenone neurolepticum which reduces stress-related increases in muscle glycolysis in pigs(14), diazepam is a benzodiazepine anxiolyticum which reduces stress effects on the autonomic nervous system(15). Piglets were then placed in hammocks(t = 15 min), and 40 mg of azaperone and 5 mg of diazepam were administered hourly during the course of the experiment. On the baseline study day (wk 1), i.v. tracers were administered to the piglets. Preceding study days in wk 2 and 3, piglets were treated with DEX, administered i.v., at a dose of 5 mg/kg, for 3 d, and also on the study day, 3 h before sedation(t = -180 min). Food intake was not affected by DEX treatment. On the study day in wk 2, one half of the piglets (n = 4), additionally received a primed, constant rate infusion of IGF-I (provided by Genentech Co., South San Francisco, CA). IGF-I was diluted in 0.9% sterile saline solution to a concentration of 100 μg/mL. Prime dose was 25 μg/kg (starting att = 30 min), followed by constant rate infusion of 25 μg/kg/h during the remainder of the experiment (t = 40 min until t= 370 min). On the study day in wk 3, the effect of DEX was determined. The other half of the piglets (n = 4) received IGF-I on the study day in wk 3, using the same regimen, after determination of the effect of DEX on the study day in wk 2. The crossover design was chosen to control for a possible influence of the total duration of DEX treatment on leucine kinetic parameters.

Exclusion criteria. Animals in which it was not possible to complete all three experiments were excluded from the study. Two piglets were excluded due to catheter obstruction before the final experiment, and one piglet due to infection.

Animals. Eight female crossbred (Landrace × Yorkshire) piglets, with an average weight of 12.5 kg (10.0-15.3 kg) on the first study day, were used for the study. Piglets were fed 50 g/kg/d of a standard diet containing 17.4 wt% protein (Hendrix B.V., Boxmeer, The Netherlands). Average weekly weight gain was 1.5 ± 0.5 kg between study days in wk 1 and wk 2, and 1.7 ± 0.7 kg between study days in wk 2 and wk 3. After an acclimatization period of 1 wk, piglets were placed under general anesthesia, and catheters (8 ch Argyle®, Sherwood Medical, Tullamore, Ireland) were placed in the right carotid artery and jugular vein, then tunneled to the back of the neck s.c. Catheters were filled with 0.9% saline solution containing 2500 U/mL heparin. Piglets were fitted with jackets to protect the catheters from unwanted removal. Hereafter, piglets were housed separately. The first experiment in each piglet was carried out 1 wk after catheter placement. Piglets were terminated after the third experiment by injection of 1 g of pentobarbital sodium. The experiment protocol was approved by the Animal Ethics Committee of the University of Groningen.

Tracer administration (see also Fig. 1). 13C-Labeled bicarbonate (99% MPE) and[L-1-13C]leucine (99% MPE) were obtained from Tracer Technologies, Inc.(Somerville, MA). Tracers were dissolved in sterile 0.9% saline solution and sterilized by filtration through a 0.22-μm filter (Millipore GMBH, Eschborn, Germany) by the hospital pharmacy. 13C-Labeled bicarbonate was administered by primed, steady-state infusion through the venous catheter(prime dose 7 μmol/kg, starting at t = 50 min, followed by infusion at a rate of 5 μmol/kg/h for 2 h, starting at t = 60 min). After 2 h, bicarbonate infusion was stopped, and 13C-labeled leucine was infused (prime dose 15 μmol/kg, starting at t = 180 min, followed by infusion at a rate of 15 μmol/kg/h for 3 h, starting att = 190 min). Tracers were administered via a calibrated infusion pump (model CS03E, MGVG, Munich, Germany).

Figure 1
figure 1

Protocol for i.v. infusions and blood sampling.13 C-Labeled tracers, as well as IGF-I, were infused i.v. at a constant rate, preceded by a prime dose (P). [13C]Bicarbonate and[1-13C]leucine were administered subsequently to determine leucine turnover and oxidation (see text for calculations). Blood samples for the determinations of isotope enrichments, blood glucose, and IGF-I levels were collected via an indwelling arterial catheter.

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Measurement of [13C]bicarbonate enrichment in arterial blood. To measure leucine oxidation, subsequent infusion of labeled bicarbonate and leucine were used, as described by Goudoever et al.(4, 16). We modified their protocol by measuring13 C-enrichment in plasma bicarbonate rather than in expired CO2. It has previously been demonstrated that 13C-enrichment in bicarbonate is equivalent to that in expired CO2 in human adults(17). To validate this assumption for our piglet model, we conducted three separate experiments in one ventilated piglet under inhalation anesthesia, using the same infusion protocol described above. During these experiments, we simultaneously collected samples of arterial blood and of expired gas to compare 13C-enrichments.13 C-Enrichment in arterial blood bicarbonate was determined as described below. For the determination of 13C-enrichment in expired CO2, 20 mL of gas were collected from the expiratory end of the ventilator, and stored in 20-mL vacuum containers (Vacutainer®, Becton Dickinson, Grenoble, France) at room temperature until analysis. A 100-μL sample of this gas was with-drawn and analyzed for 13CO2 enrichment with a Delta S isotope-ratio mass spectrometer (Finnigan MAT, Bremen, Germany). Enrichments in bicarbonate and in expired CO2 did not significantly differ from each other, the ratio of bicarbonate enrichment to that of expired CO2 was 0.97 ± 0.03 (mean ± SD,n = 26). We therefore considered it valid to measure13 C-enrichments in blood bicarbonate rather than in expired CO2.

Blood samples were taken at t = 15 and 45 min (baseline samples); at t = 90, 120, 150, and 180 min (bicarbonate plateau samples); and at t = 235, 280, 310, 340, and 370 min (leucine plateau samples), for the determination of 13C-enrichment in blood bicarbonate. Whole blood samples (2 mL) were collected via the arterial catheter, and placed in 20-mL vacuum containers (Vacutainer®, Becton Dickinson, Grenoble, France), then stored at -80°C until analyzed. Blood samples were prepared for analysis as follows: after thawing the samples in the closed vacuum containers, 1 mL of 0.2 M lactic acid was added by syringe and the sample was vortexed to quantitatively liberate CO2 from blood bicarbonate. From the headspace above the blood sample, 20 μL of gas were withdrawn and analyzed for 13CO2 enrichment with a Delta S isotope-ratio mass spectrometer (Finnigan MAT, Bremen, Germany). The coefficient of variation for measurements of [13C]bicarbonate enrichment was 5% at an enrichment of 0.005 MPE (n = 10), and 1.5% at an enrichment of 0.020 MPE(n = 10).

Measurement of [13C]KIC enrichment in plasma. To determine leucine kinetics, [13C]KIC enrichment was measured, because plasma KIC better reflects intracellular leucine enrichment than plasma leucine, and is the intracellular precursor for protein synthesis and decarboxylation(18). Blood samples were taken att = 15 and 150 min, (baseline), and at t = 235, 280, 310, 340, and 370 min for determination of [13C]KIC enrichment during[13C]leucine infusion. Heparinized blood was centrifuged immediately and plasma was stored at -80°C until derivatization. Derivatization was carried out as follows. After thawing, 150 μL of plasma were deproteinized by addition of 2.5 mL of pure ethanol and subsequent centrifugation. The supernatant was evaporated to dryness under a stream of N2 at 50°C. Then 300 μL of 1% 1,2-phenylenediamine, dissolved in 2 M hydrochloric acid, were added, and the solution was heated to 90°C for 60 min, then allowed to cool. Extraction was carried out using 3 mL of ethyl acetate, and the organic phase was evaporated to dryness under a stream of N2 at 40°C. At 15 min before analysis, 50 μL ofN,O-bis(trimethylsilyl)-trifluoroacetimide was added.[13C]KIC isotopic enrichment was measured with a model HP5995 gas chromatograph/mass spectrometer system (Hewlett-Packard Co., Palo Alto, CA). Gas chromatography was carried out on a 20 m × 0.18 mm capillary column(DB-1701, J&W Scientific, Folsom, CA). Selective ion monitoring was carried out at mass/ionic charge number (m/z) 232 for (m) and 233 for (m + 1). Plasma [13C]KIC enrichments were determined by using a calibration graph obtained by measuring weighed mixtures containing 0 to 10% MPE [13C]KIC. The coefficient of variation for determination of [13C]KIC was 3.5% (n = 10).

Blood glucose. Blood glucose was determined at t = 15, 120, 235, and 370 min, at our clinical laboratory, by a glucose analyzer based on the glucose oxidase method (Yellow Spring Inc., Yellow Springs, OH).

IGF-I. Blood samples for determination of total serum IGF-I was taken at t = 15 and at t = 370 min in all experiments, and additionally at t = 120 and at t = 235 min during IGF-I infusion. IGF-I levels were determined by specific RIA(19), after acid-ethanol extraction(20), at the Endocrinological Laboratory of the Wilhelmina Children's Hospital, University of Utrecht, The Netherlands.

Calculations. Leucine turnover was calculated according to the formula:

Leucine oxidation was calculated according to Goudoever et al.(4): Equation

Nonoxidative leucine disposal was calculated by subtracting oxidation from turnover.

Statistical analysis. Data were analyzed using repeated measures analysis of variance. When significant differences were found, comparisons were completed by t test using the Bonferroni adjustment.

RESULTS

Leucine kinetic data. Mean [13C]bicarbonate enrichments are shown in Fig. 2, mean [13C]KIC enrichments inFig. 3. The calculated leucine kinetic data are summarized in Table 1. Leucine turnover increased after DEX treatment by 18 ± 14%, compared with baseline values (p < 0.05), and also under treatment with DEX plus IGF-I versus baseline (plus 18± 8%, p < 0.01). The difference in turnover between DEX and DEX plus IGF-I was not significant. Leucine oxidation was also higher under DEX treatment by 132 ± 64% versus baseline conditions(p < 0.01), and under DEX plus IGF-I versus baseline(plus 107 ± 68%, p < 0.05). Oxidation after DEX did not significantly differ from that after DEX plus IGF-I. Non-oxidative leucine disposal was not significantly different under DEX versus baseline, DEX plus IGF-I versus baseline, or DEX versus DEX plus IGF-I.

Figure 2
figure 2

[13C]Bicarbonate enrichments. The mean[13C]bicarbonate enrichments in eight piglets under different treatment regimens are shown. Two plateaus in [13C]bicarbonate enrichment were were reached: plateau 1 during infusion of 13C-labeled bicarbonate, plateau 2 during infusion of 1-13C-labeled leucine, resulting from oxidative decarboxylation of the C1 atom of leucine. Using these plateau enrichment values, and the value for leucine turnover, leucine oxidation can be calculated(4). DEX, dexamethasone treatment,DEX/IGF-I, treatment with DEX plus IGF-I.

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Figure 3
figure 3

[13C]KIC enrichments. Mean plateau enrichments of [13C]KIC during infusion of [13C]leucine in eight piglets under different treatment regimens are shown. The lower enrichments in the DEX and in the DEX plus IGF-I group reflect increased leucine turnover rates when compared with baseline values. DEX, DEX treatment,DEX/IGF-I, treatment with DEX plus IGF-I.

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Table 1 Effect of DEX alone and combined with IGF-I on leucine kinetics in growing piglets
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Blood glucose values (Fig. 4). Blood glucose values were higher throughout under DEX treatment when compared with the baseline study. Glucose levels before starting IGF-I infusions on DEX plus IGF-I study days were similar to DEX values, but significantly lower than under DEX during infusion of IGF-I. At t = 120 and 370 min, glucose values did not significantly differ between DEX plus IGF treatment and baseline conditions.

Figure 4
figure 4

Blood glucose values. In animals treated with DEX only, glucose levels were increased when compared with baseline conditions. When IGF-I infusion (starting at t = 30 min) was added to DEX treatment, glucose levels decreased and were not significantly different from baseline values at t = 235 and at t = 370 min. DEX, DEX treatment, DEX/IGF-I, treatment with DEX plus IGF-I.

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IGF-I values (Fig. 5). Serum IGF-I levels at the start of the experiment were not significantly different between treatment groups. In the DEX plus IGF-I group, IGF-I levels after starting IGF-I infusion increased rapidly. In the baseline experiments, IGF-I levels were slightly higher at t = 370 min when compared with the onset of the experiment.

Figure 5
figure 5

Serum IGF-I. Total serum IGF-I did not significantly differ between the three treatment groups at the onset of the experiment. In DEX-treated animals receiving an infusion of IGF-I (starting at t = 30 min), values more than doubled when compared with baseline values.DEX, DEX treatment; DEX/IGF-I, treatment with DEX plus IGF-I.

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DISCUSSION

Our study shows that IGF-I does not reverse corticosteroid-induced protein catabolism in growing piglets. To investigate corticosteroid-induced protein catabolism, we treated piglets with the corticosteroid DEX and used[13C]leucine kinetics to measure protein metabolism. In a dose-finding study (data not shown), DEX, at a dose of 5 mg/kg/h, administered for 4 d, was found to produce changes in leucine kinetics in piglets similar to those reported in corticosteroid-treated human adults(2) and infants(4). In our study, DEX increased leucine turnover and oxidation, indicating whole body protein catabolism. Adding IGF-I to DEX treatment failed to reverse these changes in leucine kinetics.

There are several possible explanations for the failure of IGF-I to reverse corticosteroid-induced protein catabolism in piglets. One is that IGF-I does not mediate all actions of GH. Assuming that GH reverses corticosteroid-induced protein catabolism, as has been shown in human adults(8) and in rats(7, 21), GH could mediate this reversal via an IGF-I independent mechanism(22), or by stimulating paracrine secretion of IGF-I(23), or by modifying IGF-I action through effects on IGF binding protein production(24). None of these mechanisms of GH action are reproduced when IGF-I is infused(22, 24). Another possible explanation is that the GH/IGF-I axis may not yet be physiologically active in very young animals. The anabolic response to GH increases with age in growing pigs(25). Data are lacking on whether GH has any anabolic effect in piglets of the size and age used in our study. Further evidence for the age dependence of the activity of the GH/IGF-I axis is provided by the fact that the somatogenic GH receptor is absent in the ovine fetal liver and does not appear until after birth(26). In a recent study in small-for-date premature infants, GH failed to produce a protein anabolic effect(27). A third explanation for the lack of effect of IGF-I could be a specific disturbance of IGF-I action through DEX. In human volunteers, DEX decreases IGF-I bioactivity, even in the presence of increased immunoreactive IGF-I levels(28). In fasted lambs(9) and in calorically restricted human volunteers(29), IGF-I decreases leucine oxidation in a dose-dependent manner, suggesting IGF-I may decrease protein catabolism in catabolic states not involving corticosteroid treatment. The final explanation is that the dose of IGF-I administered in our study could have been insufficient to cause a protein anabolic effect. IGF-I has been shown to reduce whole body protein catabolism in DEX-treated rats(30–32). In these studies very high doses of IGF-I (690 μg/kg/h) were used to achieve an anabolic effect. At doses similar to those used in our study, no protein anabolic effect was observed. The only study that we are aware of addressing the role of IGF-I in corticosteroid-induced protein catabolism in humans(22) failed to show a protein anabolic effect of IGF-I. However, a low dose was used (5-10 μg/kg/h). In our study, IGF-I at a dose of 25 μg/kg/h decreased serum glucose levels to baseline values. In view of the risk of hypoglycemia at higher IGF-I doses(10, 33), we considered it inadvisable to increase the dose of IGF-I in our study.

Our study further shows that IGF-I attenuates corticosteroid-induced hyperglycemia. The corticosteroid DEX increased blood glucose levels nearly 2-fold over baseline values. After adding IGF-I to DEX, blood glucose values decreased to near baseline values. This is probably due to cross-reaction of IGF-I with the insulin receptor(22). However, the lowering of blood glucose levels alone, without beneficial effect of IGF-I on protein metabolism, would not warrant treatment with IGF-I.

We conclude that IGF-I does not reverse corticosteroid-induced protein catabolism in growing piglets. IGF-I attenuates corticosteroid-induced hyperglycemia. We suggest that IGF-I may not be of therapeutic value in corticosteroid-induced protein catabolism in pediatric patients.