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Effect of Short-Term Prednisone Use on Blood Flow, Muscle Protein Metabolism, and Function

Endocrinology Research Unit, Mayo Clinic School of Medicine, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: K. S. Nair, M.D., Ph.D., Mayo Clinic School of Medicine, Endocrinology Research Unit, 5-194 Jo, 200 First Street SW, Rochester, Minnesota 55905. E-mail: nair.sree{at}mayo.edu.

	Abstract

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Glucocorticoids can cause muscle atrophy, but the effect on muscle protein metabolism in humans has not been adequately studied to know whether protein synthesis, breakdown, or both are altered. We tested the effect of 6 d of oral prednisone (Pred, 0.5 mg/kg·d) on muscle protein metabolism and function. Six healthy subjects (three men/three women, 22–41 yr) completed two trials (randomized, double-blind, cross-over) with Pred and placebo. Fasting glucose, insulin, IGF-I, and glucagon were higher on Pred vs. placebo, whereas IGF-II and IGF binding protein-1 and -2 were lower. Whole-body amino acid fluxes, blood urea nitrogen, and urinary nitrogen loss were not statistically different between trials. Leg blood flow was 25% lower on Pred leading to 15–30% lower amino acid flux among the artery, vein, and muscle. However, amino acid net balance and rates of protein synthesis and breakdown were unchanged, as were synthesis rates of total mixed, mitochondrial, sarcoplasmic, and myosin heavy chain muscle proteins. Muscle mitochondrial function, muscle strength, and resting energy expenditure were also unchanged. These results demonstrate that a short-term moderate dose of prednisone affects glucose metabolism but has no effect on whole-body or leg muscle protein metabolism or muscle function.

	Introduction

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GLUCOCORTICOIDS ARE EXTENSIVELY used to treat clinical conditions such as inflammation, asthma, and immune suppression. However, there are undesirable effects that arise in the presence of excess glucocorticoids, including inhibition of insulin action on glucose metabolism (1, 2, 3, 4) and muscle wasting and weakness (5, 6, 7). A reduction in muscle mass implies that glucocorticoids alter the balance between protein synthesis and breakdown, although the exact mechanisms have not been fully clarified.

Studies in rodents have shown that high-dose glucocorticoid administration increases protein breakdown in skeletal muscle by enhancing the expression and activity of components of protein catabolism pathways (8, 9, 10, 11). There is also evidence that muscle protein synthesis is suppressed and that this is due, at least in part, to inhibition of the complexes involved in initiation of protein translation (12, 13, 14, 15, 16). Whereas these studies have been useful for identifying the potential events that lead to muscle wasting, their applicability to humans is limited by the fact that the doses of glucocorticoids given experimentally to rats are much higher than would typically be administered to humans. Furthermore, some of the effects attributed to glucocorticoids, such as the dramatic loss of body and muscle weight, may be due in part to the anorexic effect that occurs in rodents in response to these high doses (12, 17, 18).

Nearly all of the previous human studies examining the effect of glucocorticoids on protein metabolism have been limited to measurements of whole-body amino acid kinetics in young healthy people after short-term glucocorticoid administration. It was observed in some of those studies that protein breakdown, as assessed by the appearance rate of leucine (Leu) or phenylalanine (Phe) using amino acid tracers, is increased after 6–7 d of moderate- to high-dose (0.5–0.8 mg/kg·d) prednisone administration in healthy volunteers (19, 20, 21, 22). Whole-body oxidation of Leu may also be increased by glucocorticoids (19, 20, 21, 22), whereas whole-body protein synthesis is typically unchanged (19, 20, 22) or slightly decreased (21). However, because whole-body studies represent the average protein turnover of all of the body protein pools, it is not possible to determine whether the results reflect changes in individual tissues, such as skeletal muscle.

There are limited data available on the effect of glucocorticoids on human muscle protein metabolism. Beaufrere et al. (19) measured the appearance of urinary 3-methyl histidine, an index of muscle protein breakdown, and observed no change between prednisone (0.8 mg/kg·d for 5 d) and placebo trials. This led the authors to conclude muscle protein breakdown was not affected by short-term glucocorticoid elevation. However, because nonmuscle tissues, such as the gut, can produce 3-methyl histidine, this measure may not be sufficiently specific and sensitive to detect the treatment effect in muscle. This observation also does not rule out the possibility that glucocorticoids may have an effect on muscle protein synthesis rate. In two other studies, arteriovenous amino acid balance across the forearm was examined in young healthy people after 4 d of oral dexamethasone (8 mg/d), which has higher potency than prednisone (3, 4). In those studies, the authors pointed to a nonstatistically significant trend for more negative net balance of Phe in the fasted state as evidence that glucocorticoid use resulted in greater tissue protein loss. However, neither amino acid uptake (a marker of protein synthesis) nor appearance (from protein breakdown) across the forearm was significantly altered by glucocorticoid treatment (3, 4). Only one study (23) has directly measured muscle protein synthesis rate using muscle biopsy methods in humans. In that study, women (mean age 58–71 yr) with rheumatoid arthritis undergoing knee surgery who had used prednisone for 9 yr had a 30% lower rate of synthesis of mixed (total) muscle proteins in the quadriceps muscle, compared with arthritis patients who had not used corticosteroids (23). It is unclear, however, how much the disease status, physical activity history, and surgical treatment of these patients contributed to the observed effects. This finding requires confirmation under well-controlled conditions. Thus, at the present time, there are no studies that have simultaneously used arteriovenous balance and muscle biopsy methods to comprehensively determine whether excess glucocorticoids alter protein synthesis and/or breakdown in human skeletal muscle.

In the current investigation, we tested whether short-term (6 d) administration of glucocorticoids would alter muscle protein metabolism in healthy subjects. We used these experimental conditions to avoid confounding factors that might arise if the studies were performed in patients with disease and so that our data would be comparable with previous human studies. To perform a more comprehensive evaluation of protein metabolism than prior work in this area, amino acid kinetics were measured at the level of the whole body and across the leg, using arteriovenous balance techniques. Muscle biopsies of the vastus lateralis were also obtained to measure the fractional synthesis rates of muscle proteins and the oxidative capacity of the tissue. Results were confirmed using multiple amino acid tracers.

	Subjects and Methods

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Materials

L-[1,2-¹³C]Leu (97 atom percent excess) was purchased from Mass Trace (Woburn, MA) and Isotec Inc. (Miamisburg, OH). L-[¹⁵N]Phe (97 atom percent excess), L-[¹⁵N]tyrosine (Tyr, 97 atom percent excess), and [²H₄]Tyr (91 atom percent excess) were purchased from Cambridge Isotope Laboratories, Inc. (Woburn, MA). Isotopes were tested before use for their isotopic and chemical purity. The isotope solutions were prepared under sterile conditions and were determined to be bacteria and pyrogen free before their administration to humans. Luciferin/luciferase reagent for ATP monitoring (formula SL) was purchased from BioThema (Haninge, Sweden) and ADP and ATP from Roche Molecular Biochemicals (Indianapolis, IN). All other reagents for mitochondrial assays were purchased from Sigma Chemicals (St. Louis, MO). The study protocol was approved by the Institutional Review Board of Mayo Foundation. All procedures were performed in accordance with the ethical guidelines of the Declaration of Helsinki and were clearly explained to the study volunteers during their initial visit. Each participant provided his or her informed oral and written consent before enrollment into the study.

Participants

Six young, healthy people (three men, three women) volunteered to participate in the study after responding to advertisements placed in the local area (Rochester, MN). Average characteristics (mean ± SEM) of the group were: age 30 ± 3 yr, height 173 ± 2 cm, weight 72.6 ± 3.5 kg, body mass index 24.2 ± 1.0 kg/m², body fat-free mass 51.1 ± 3.9 kg, and body fat 24.4 ± 3.4%. Body composition was determined using dual-energy x-ray absorptiometry. Health status was assessed by medical history, physical exam, blood chemistries (including liver enzymes, creatinine, electrolytes, and glucose), complete blood count, urinalysis, and electrocardiogram. Inclusion criteria included age (18–45 yr) and body mass index (20–28 kg/m²). Exclusion criteria included tobacco use, ß-blockers or any medications that could affect metabolism or blood coagulation, diabetes or other endocrine disorders, and debilitating chronic illness. None of the participants were taking medications at the time of the study, nor were they engaged in a regular exercise program.

Protocol and procedures

Each participant completed two similar trials separated by an average of 6 wk (range 5–8 wk). Regular lifestyle patterns were maintained between trials. During each study period, either prednisone or placebo was administered in a randomized, double-blind manner for 6 d. Capsules containing prednisone (0.5 mg/kg·d) or placebo were consumed each morning with food for the first 5 d. The same dose was consumed on the sixth day without food. The capsules were prepared by the Mayo Pharmacy Department and were indistinguishable from each other. During each study period, a weight-maintaining diet (55:30:15% carbohydrate, fat, and protein, respectively) was provided on d 3–5 of the treatment. Strenuous physical activity was avoided on d 3–5. On the morning of d 5, muscle strength testing was performed, as described below. That evening (1800 h), participants were admitted to the General Clinical Research Center (GCRC) for in-patient study. A light snack was provided at 2200 h, and no food was consumed thereafter until completion of the study the next day.

The following morning (d 6), the last dose of prednisone or placebo was taken at approximately 0530 h. Within the next hour, a polyethylene venous catheter was placed in an antecubital arm vein for infusion of isotopic tracers. Primed, continuous infusions of [1,2-¹³C]Leu (10.4 µmol/kg prime, 10.4 µmol/kg·h thereafter), [¹⁵N]Phe (4.2 µmol/kg prime, 4.2 µmol/kg·h thereafter), [²H₄]Tyr (3.0 µmol/kg prime, 3.0 µmol/kg·h thereafter), and [¹⁵N]Tyr (1.6 µmol/kg prime only) were maintained for 8 h. The start of the infusion is designated as time 0 min. Once the infusion of tracers was started, subjects were transported a short distance to the Vascular Radiology Laboratory for placement of lines in the femoral artery and vein for infusion and sample collection (24, 25). French sheaths were inserted into the femoral artery and vein of the right leg on the first trial and in the left leg on the second trial. A femoral artery catheter was inserted through the arterial sheath with the catheter tip in the common iliac artery. This catheter was used for arterial blood sampling, and the sheath was used to infuse indocyanine green. The distal tip of the venous sheath was placed in the external iliac vein a few centimeters above the inguinal ligament. The volunteers were then transferred back to the GCRC for completion of the study. The arterial and venous lines were maintained by normal saline infusion.

Leg blood flow was determined by indicator-dilution technique during arterial infusion of indocyanine green from 120 to 210 min and again from 390 to 480 min (24, 25). Blood samples were drawn from the femoral artery and vein at 150, 170, 190, and 210 min and again at 420, 440, 460, and 480 min. Muscle biopsies of the vastus lateralis were obtained under local anesthesia at 240 and 480 min of tracer infusion (26). The biopsies were performed on the same leg that was catheterized, and the second biopsy site was approximately 8–10 cm proximal from the first biopsy. A portion of the muscle was kept on ice in saline-soaked gauze for mitochondrial studies, as described below. The remainder of the tissue was rapidly frozen in liquid nitrogen and stored at –80 C. Resting energy expenditure was determined by indirect calorimetry (DeltaTrac, SensorMedics, Yorba Linda, CA) for 45 min beginning at approximately 270 min. The last 30 min of this measurement were used for data analysis. Upon completion of the study, leg catheters were removed and participants remained overnight for observation before being discharged from the GCRC.

Hormone and metabolite assays

Glucose was measured with a Beckman glucose analyzer (Beckman Instruments, Porterville, CA). Nonesterified free fatty acids were measured using an enzymatic colorimetric assay (NEFA C; Wako Chemicals USA, Richmond, VA). Plasma levels of amino acids were measured by an HPLC system (HP 1090, 1046 fluorescence detector and cooling system) with precolumn O-phthalaldehyde derivatization (27). Urinary nitrogen content was measured using a Beckman GM7 Analox Microstat (Beckman Instruments).

Insulin and human GH were measured with two-site immunoenzymatic assays (Access system, Beckman Instruments, Chaska, MN). Glucagon was measured by a direct, double-antibody RIA (Linco Research, St. Louis, MO).

After separation from their binding proteins with a simple organic solvent, total IGF-I and IGF-II were measured with two-site immunoradiometric assays (Diagnostic Systems Laboratories, Webster, TX). IGF binding protein (IGFBP)-1 and -3 were also measured with two-site immunoradiometric assays, whereas IGFBP-2 was measured by a double-antibody RIA (Diagnostic Systems Laboratories).

Plasma amino acid kinetics

The enrichment level of [1,2-¹³C]Leu in plasma was determined using a gas chromatograph/mass spectrometer (GC/MS; HP5973, Hewlett-Packard Instruments, Avondale, CA) by multiple ion monitoring at m/z 342/344 under positive ion methane chemical ionization conditions. The concentration of L-Leu was simultaneously determined by comparison with a norleucine internal standard. [¹⁵N]Phe, [¹⁵N]Tyr, and [²H₄]Tyr were measured as their t-butyldimethylsilyl ester derivatives under electron ionization conditions using a gas chromatograph/mass spectrometer (Voyager, Finigan MAT, Bremen, Germany). Fragment ions were monitored at m/z 345/337/336 for Phe and m/z 472/470/467/466 for Tyr. [1,2-¹³C]Ketoisocaproate (KIC) in plasma was determined as its quinoxalinol-trimethylsilyl ether derivative under electron ionization conditions using an HP5988 GC/MS (Hewlett-Packard) (28). KIC concentration was measured simultaneously in the same samples by comparison with ketoisovalerate, which was added as an internal standard. All samples were analyzed in duplicate.

For calculation of whole-body amino acid kinetics, the mean values of isotopic enrichment from 3 to 8 h of infusion were used. Whole-body flux rates of Leu, Phe, and Tyr were calculated by tracer dilution using the equation, Q = i[(Ei/Ep) – 1], where Q represents flux of a particular amino acid, i is the rate of tracer infusion, and Ei and Ep are the enrichment of the tracer in the infusate and the plasma at isotopic plateau, respectively. For Tyr flux the enrichment of [²H₄]Tyr was used. The Phe conversion to Tyr (Qpt) was calculated as previously reported (29, 30). The Phe incorporation into protein (Sp) for whole body is calculated by subtracting Qpt from Qp because Phe is either irreversibly converted into Tyr or incorporated into protein (29, 30).

Calculation of amino acid kinetics across the leg was performed using two methods. The first method used arterial and venous amino acid concentration and enrichment and a measure of blood flow (24, 25). This yielded estimates of net concentration balance and the rate of appearance (Ra) and disappearance (Rd) of a given amino acid. Ra represents amino acids appearing into the circulation from protein breakdown, whereas Rd is a measure of amino acids leaving the circulation into tissue. The second calculation was a three-pool model that also used arterial and venous amino acid concentration and enrichment and blood flow as well as a measure of the intracellular enrichment of the tracer in the tissue of interest, which in this case was skeletal muscle (31). Although the model was originally developed for use with muscle tissue fluid (free amino acid pool), it has been recently demonstrated that the derived flux values are significantly different from flux values calculated using enrichment in the amino-acyl tRNA pool (31). The advantage of using amino-acyl tRNA for this purpose is that it is assumed to reflect the tracer enrichment in the immediate precursor pool for protein synthesis, whereas the tissue fluid pool is a mixture of both intracellular (85%) and extracellular (15%) free amino acids. Therefore in the current study, we used amino-acyl tRNA for these calculations.

Muscle protein synthesis

A 150-mg portion of each muscle sample was used for the isolation of mitochondrial and sarcoplasmic protein fractions by differential centrifugation as previously described (26, 32, 33). A separate 20- to 30-mg piece of muscle was used to prepare total mixed muscle proteins and isolate free tissue fluid amino acids (34). Amino-acyl tRNA was isolated from a 150-mg piece of muscle (34).

The muscle protein fractions were hydrolyzed overnight in 0.6 M HCl in the presence of cation exchange resin (AG-50, Bio-Rad Laboratories, Hercules, CA) and purified the next day using a column of the same resin. The amino acids were dried (SpeedVac, Savant Instruments, Hicksville, NY) and then derivitized as their trimethyl acetyl methyl ester. [¹³C]Leu and [¹⁵N]Phe enrichments in muscle proteins were determined using a gas chromatograph-combustion-isotope ratio mass spectrometer (Delta Plus, Finigan MAT) as described previously (35, 36). Tissue fluid amino acids and amino-acyl tRNA samples were derivatized as their t-butyldimethylsilyl ester and analyzed for [¹³C]Leu and [¹⁵N]Phe enrichments using a GC/MS (HP5973, Hewlett-Packard Instruments) under electron ionization conditions (34, 35, 37).

The fractional synthetic rates of mitochondrial and sarcoplasmic proteins were calculated using the equation,

where (E8 h – E3 h) represents the increment in [¹³C]Leu or [¹⁵N]Phe enrichment in muscle proteins between 3 and 8 h of infusion. Ep is the average precursor pool enrichment of [¹³C]Leu or [¹⁵N]Phe in either muscle tissue fluid or amino-acyl tRNA taken from the 3- and 8-h biopsies. T is the time of incorporation between the two biopsies, which in this case was 5 h.

Muscle oxidative capacity

Mitochondria were isolated by centrifugation from fresh muscle tissue, and ATP production capacity was assessed using a bioluminescent method as previously described (38, 39). Briefly, mitochondria were added to cuvettes containing luciferin, luciferase, 0.3 mM ADP, and one of six substrate combinations. Substrates used were, in mM, 10 glutamate + 1 malate, 10 -ketoglutarate, 1 pyruvate + 1 malate, 0.05 palmitoyl-L-carnitine + 1 malate, 20 succinate + 0.1 rotenone, or 1 pyruvate + 0.05 palmitoyl-L-carnitine + 10 -ketoglutarate + malate. ATP production was measured simultaneously for all reactions in triplicate at 25 C in BioOrbit 1251 luminometer (BioOrbit Oy, Turku, Finland). Each reaction was calibrated using an internal ATP standard. A separate piece of muscle (20 mg) was homogenized as a buffer containing 20 mM HEPES, 1 mM EDTA, and 250 mM sucrose (pH 7.4), supplemented with a protease inhibitor cocktail (Complete Mini, Roche Applied Science, Indianapolis, IN). Aliquots of the homogenate were used to measure protein concentration (DC protein assay, Bio-Rad Laboratories) and the activity of the mitochondrial enzymes citrate synthase (CS, from the Krebs cycle), cytochrome c oxidase (COX, part of the respiratory chain), and L-3-hydroxyacyl coenzyme A dehydrogenase (HAD, a step in fatty acid ß-oxidation) using spectrophotometric assays at 25 C (32, 39, 40).

Muscle strength tests

Three tests of upper-body strength were conducted on the morning of d 5 of each study phase. Isometric handgrip strength was determined from a series of six maximal efforts. The best of the six trials was taken for data analysis. Chest press and arm (biceps) curl strength were measured as the one-repetition maximum weight lifted during a progressive series of attempts. Two familiarization sessions were completed approximately 1 and 2 wk before commencing the study. This assured that the subjects could reliably generate maximal efforts with a minimal number of attempts. No lower-body exercises were performed to minimize the chance that muscle activation would affect protein metabolism of the legs on the following day.

Statistical analysis

Summarized values are reported as mean ± SEM. Paired t tests were used for comparisons between placebo and prednisone trials, with -level set to 5% to define statistical significance.

	Results

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Plasma metabolites and hormones

Compared with the placebo condition, prednisone resulted in statistically increased levels of circulating glucose, insulin, C-peptide, glucagon, and IGF-I (Table 1). There was also a trend for increased nonesterified fatty acids (P = 0.068) during the prednisone trial. During the prednisone trial, levels of IGF-II, IGFBP-1, and IGFBP-2 were significantly reduced, whereas GH and IGFBP-3 levels were not statistically different from the placebo trial. There were no statistical differences between trials in the concentration of any plasma amino acids or KIC (Table 2).

In the overnight fasting state, the respiratory exchange ratio was 0.76 ± 0.02 during the placebo trial and 0.75 ± 0.02 during the prednisone trial (P = 0.908), indicating that substrate use between trials was similar. Likewise, there were no significant differences between trials in resting oxygen consumption (257 ± 16 vs. 281 ± 22 ml/min for placebo and prednisone, respectively, P = 0.168) or resting metabolic rate (73 ± 5 vs. 80 ± 6 kcal/h for placebo and prednisone, respectively, P = 0.184).

Protein kinetics and leg blood flow

Blood urea nitrogen levels were not statistically different between trials [13 ± 2 mg/dl (4.5 ± 0.6 mmol/liter) for placebo and14 ± 2 mg/dl (4.9 ± 0.8 mmol/liter) for prednisone, P = 0.493]. Urinary nitrogen loss during the study day tended to be higher during the prednisone trial [0.22 ± 0.04 g/h (16 ± 3 mmol/h)] than during placebo [0.17 ± 0.03 g/h (12 ± 5 mmol/h)], but the difference was not statistically significant (P = 0.111). Leg blood flow while on prednisone (33 ± 6 ml/min·kg leg fat-free mass) was 25% lower (P = 0.060) than during the placebo trial (45 ± 5 ml/min·kg leg fat-free mass). Enrichment of the free amino acid pools in plasma and muscle as well as the amino-acyl tRNA and protein-bound enrichments in muscle are shown in Table 3.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Whole-body amino acid kinetics. Qphe, Qtyr, and Qleu, Flux rates of Phe, Tyr, and Leu, respectively; Qpt, rate of conversion of Phe to Tyr; Sp, incorporation of Phe into protein; FFM, fat-free mass. Paired t tests comparisons between treatments all had P > 0.30.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Amino acid kinetics across the leg using standard dilution equations (24 25 ). Net balance of Leu, Phe, and Tyr were all negative, indicating a net release of amino acids during the postabsorptive state (top). The Ra and Rd for Leu, Phe, and Tyr are shown in the bottom panel. Paired t test comparisons between treatments all had P > 0.45. FFM, Fat-free mass.

View larger version (25K): [in this window] [in a new window]   FIG. 3. Leg amino acid kinetics determined from a three-pool model with amino-acyl tRNA enrichment in tissue (31 ). Flux rates are given for the inward and outward fluxes in the leg (Fin and Fout, respectively), and flux from artery to vein (Fva), tissue to vein (Fvt), and artery to tissue (Fta). PB and PS are protein breakdown and synthesis, respectively. Numbers over bars are P values for paired t test comparisons between treatments. FFM, Fat-free mass.

View larger version (26K): [in this window] [in a new window]   FIG. 4. Synthesis rates of muscle proteins. Rates are shown for total mixed muscle proteins and the mitochondrial (Mito) and sarcoplasmic (Sarco) subfractions using either Leu (top) or Phe (tracers). Rates were calculated using either the muscle tissue fluid (left) or amino-acyl tRNA (right) enrichment as the precursor pool. Paired t test comparisons between treatments all had P values > 0.20.

Activity of each of the mitochondrial oxidative enzymes measured (CS, COX, and HAD) was not statistically different between treatments (Table 4). There were also no differences between treatments for mitochondrial ATP production with all but one substrate combination. The exception was that there was a small (12%) but statistically significant increase in ATP production with palmitoyl-L-carnitine and malate during the prednisone trial vs. placebo.

	Discussion

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The results of the current study demonstrate that postabsorptive whole-body and muscle protein metabolism is not adversely affected by 6 d of oral prednisone administration at a dose of 0.5 mg/kg·d in healthy people. Muscle strength and all but one index of muscle oxidative capacity were also unaffected. In contrast, postabsorptive levels of plasma glucose and several hormones were significantly altered and leg blood flow was reduced. This suggests that carbohydrate metabolism and other endocrine systems are relatively more sensitive to the effects of glucocorticoids than whole-body and muscle protein metabolism.

We hypothesized that prednisone use would result in higher muscle protein breakdown and/or reduced muscle protein synthesis. There was a strong trend for leg blood flow to be lower during the prednisone trial, which may account for the modest changes in flux rates among the femoral artery, femoral vein, and leg muscle tissue as calculated by the three-pool model (Fig. 3). To our knowledge, the present study is the first to report a reduction in leg blood flow in humans in response to glucocorticoids. The reduction in leg blood flow occurred in five of the six participants (the sixth had no change). In two previous studies conducted after 4 d of dexamethasone administration, there were no statistically significant changes in forearm blood flow detected in the basal state (3, 4), although there was a trend for a 20% reduction in flow during the dexamethasone trial in one of those reports (3). Recent work in pigs revealed that a single pharmacological dose of prednisone results in reductions in blood flow to the muscle, skin, and bone in the hip area that are evident within 1 h and persist at least 24 h (41). This rapid onset of effect suggests that the blood flow reduction arises from so-called nongenomic effects of glucocorticoids that are not mediated through transcription or translation. A potential mechanism for the effect on blood flow was revealed by a recent study that showed that glucocorticoids have a detrimental effect on vascular epithelial cells (42). In that study human umbilical vein epithelial cells exposed to dexamethasone produced less nitric oxide, apparently due to higher presence of oxidants such as hydrogen peroxide and peroxynitrite. Thus, those authors proposed that reduced nitric oxide production could prevent vasorelaxation leading to reduced blood flow as well as higher risk of vascular complications for long-term glucocorticoid users (42).

Despite the lower blood flow that led to reduced amino acid movement through the leg, there was no change in protein breakdown or synthesis detected using either the Leu or Phe tracer or with the different methods of calculation. There was also no change in the fraction synthesis rate of mixed (total) muscle proteins, or the subfractions of mitochondrial, sarcoplasmic, or myosin heavy-chain proteins. The effect of glucocorticoid administration on fractional synthesis rate of muscle proteins humans has not been previously examined under well-controlled experimental conditions. Comparisons with animal studies are problematic because much higher doses have typically been used in rodents. Nevertheless, previous studies in rats have shown that high-dose administration of glucocorticoids for 5–12 d results in pronounced skeletal muscle atrophy (up to 50% reduction in some muscles) and is accompanied by a reduced rate of synthesis of total mixed muscle proteins and myosin heavy chain (15, 16). These effects in rodents have been shown to be more prominent in fast-twitch, glycolytic muscles (i.e. plantaris, gastrocnemius) than in oxidative muscles (i.e. soleus), although the mechanism for such tissue specificity is not yet known (15, 16).

In previous investigations in humans, whole-body protein breakdown in the postabsorptive state was increased by short-term glucocorticoid use (3, 19, 20, 21, 22). In those studies whole-body Leu oxidation was also increased and whole-body protein synthesis was either unchanged or decreased (19, 20, 21, 22). Thus, the balance of amino acid metabolism was shifted in favor of a more catabolic state by glucocorticoids, and this was supported by increased loss of urinary nitrogen (3, 21, 22). Surprisingly, however, it was reported that whole-body amino acid kinetics were not altered in patients with Cushing’s syndrome (43). This latter finding requires confirmation in specific studies examining skeletal muscle metabolism because it is inconsistent with the loss of protein mass in these patients. Unlike those earlier reports, we did not detect a significant alteration in whole-body protein breakdown after prednisone administration. The reason for this discrepancy as well as the lack of prednisone effects on muscle protein turnover is not yet clear. However, the strength of the current investigation was that the study outcomes were confirmed with multiple amino acid tracers at the whole-body, arteriovenous, and muscle protein levels using some of the most detailed techniques currently available. We used both a compartmental analysis model recently developed to measure leg muscle protein kinetics (31) as well as direct measurement of the fractional synthesis rate of muscle proteins using amino acyl t-RNA as the precursor pool. Blood urea nitrogen and urinary nitrogen losses were also not significantly altered. Thus, several independent measurements corroborate the lack of effect of prednisone on whole-body and muscle protein kinetics in this study.

The dose and duration of prednisone administered in the current study (0.5 mg/kg·d for 6 d) was at the lower range of what has been used in previous investigations of protein metabolism in healthy human volunteers, with doses of approximately 0.5 (21, 22) or 0.8 mg prednisone/kg·d (1, 19, 20) given for 5–7 d. Dexamethasone, which has higher potency than prednisone, was given orally at 8 mg/d for 4 d in two other investigations (3, 4). It is clinically established that long-term glucocorticoid excess is associated with muscle wasting and weakness (5, 6, 7), and it was reported that prednisolone use for an average of 9 yr at 8 mg/d to treat rheumatoid arthritis was associated with reduced rate of synthesis of muscle proteins (23). We therefore propose that either a higher dose or longer duration of prednisone administration than was used in the current study is required to alter postabsorptive protein metabolism in young healthy people. This is line with rodent studies in which high doses of glucocorticoids have been used to demonstrate large rapid effects on protein synthesis and breakdown (8, 9, 10, 11, 12, 13, 14, 15, 16). However, our aim was to use a dose and duration scheme that would be relevant to the common clinical use of glucocorticoids for short-term treatment of conditions such as inflammation or asthma. The present study results indicate that there are no apparent disturbances in muscle protein metabolism, at least in young healthy people. The threshold for glucocorticoid effects may differ with age or health status. For example, it was reported that dexamethasone has more deleterious effects on older rats (18 months), compared with younger rats (6–8 months) (10). The effect of glucocorticoids on protein metabolism may also be less evident in the postabsorptive state, compared with after a meal. Beaufrere et al. (19) showed that following a gastrically infused meal, prednisone prevented the normal increase in whole-body net balance of protein, apparently due to higher postmeal oxidation of Leu. The elevation of glucose, insulin, C-peptide, and glucagon in the present study suggests that carbohydrate metabolism may be more likely to be impaired by glucocorticoids in the postabsorptive state than protein metabolism (1, 2, 3, 4).

Prednisone use resulted in elevated insulin and IGF-I levels. The rise in these two hormones may provide an anabolic stimulus to counteract the glucocorticoid effect on protein metabolism. A major action of insulin on protein metabolism is suppression of muscle protein breakdown (24, 25). Thus, elevated insulin levels during prednisone administration may have prevented an increase in protein breakdown in the current study. This possibility is supported by reports that insulin action on protein is maintained in both short-term prednisone users and patients with Cushing’s syndrome (21, 22, 43). Arguing against this possibility, however, is the fact that short-term glucocorticoid use can increase the rate of whole-body protein catabolism, whereas circulating insulin is also elevated (3, 4, 19, 21). There is also evidence that glucocorticoids may actually blunt the ability of insulin to suppress protein breakdown (1, 3). Thus, the interplay between insulin and glucocorticoids is not yet sufficiently resolved to reliably determine whether the rise in circulating insulin could have prevented some or all of the predicted effects of glucocorticoids in the current study.

Likewise, IGF-I has been reported to prevent the effects of prednisone on protein metabolism when coadministered (100 µg/kg·d) with prednisone (0.8 mg/kg·d) for 5 d in young healthy subjects (44). A lower dose of IGF-I (80 µg/kg·d) used in another study, however, did not effectively alter the prednisone effect on protein metabolism (22). This suggests that there may be a minimal level of IGF-I required to counter the glucocorticoid effects on protein. Subjects in the current study demonstrated an average increase of 35% in plasma IGF-I after prednisone administration, which is greater than previously reported under similar treatment conditions (20, 21, 22). Because the levels of IGFBP-1 and IGFBP-2 were decreased by 44 and 13%, respectively, in the prednisone trial, the bioavailability of IGF-I could be further increased. Thus, the elevated IGF-I may play a counterregulatory role to maintain protein turnover at normal levels. The 6% reduction in circulating IGF-II in the prednisone trial could potentially dampen this stimulus, although the change in IGF-II is small in comparison with the increase in IGF-I. Furthermore, IGF-II has been shown to play an important role in skeletal muscle development, i.e. proliferation and differentiation, but its effects on muscle metabolism during adulthood have not been established (45, 46).

The large (44%) decline in circulating IGFBP-1 level during the prednisone trial is most likely due to the increase in insulin, which has been shown to be a potent inhibitor of IGFBP-1 production (47). Previous studies reported that glucagon can stimulate production of IGFBP-1 (48, 49). However, in the prednisone trial of the present study, the increase in glucagon (12%) was much smaller than the increase in insulin (109%), so any stimulatory effect of glucagon would have been negated by the larger inhibitory effect of insulin. Thus, insulin action (specifically on glucose metabolism) appears to be diminished by prednisone treatment, but the compensatory increase in insulin seems to reduce IGFBP-1 levels. Consistent with our findings, Miell et al. (50) reported that after 3 d of treatment with dexamethasone (4 mg/d), normal male volunteers had increased levels of insulin and IGF-I and decreased levels of IGFBP-1 and IGFBP-2. In that study, though, there was also an increase in IGFBP-3 whereas IGF-II was unchanged, both of which differ from our results. Collectively the data suggest that changes in the IGF system during glucocorticoid treatment are mostly responses to the changing metabolic condition rather than direct effects of glucocorticoids (47).

Finally, there were almost no changes in muscle function in response to the short-term use of prednisone because muscle strength and nearly all of the measures of muscle mitochondrial function were unchanged. The one exception was that mitochondrial ATP production in the presence of palmitoyl-L-carnitine was increased 12% during the prednisone trial, which suggests that the capacity to oxidize fatty acids for fuel production in muscle was increased. To our knowledge such a finding has not been previously reported. Prednisone had no significant effect on the activity of HAD, a key enzyme in the ß-oxidation pathway. The other mitochondrial enzymes and ATP production measurements tested, which are part of or share the common pathways of the Krebs cycle and respiratory chain, were also unchanged. Thus, at least one other unique step in fatty acid transport or oxidation was affected by prednisone to cause the increase in ATP production with palmitoyl-L-carnitine. Carnitine palmitoyl transferase is a likely candidate because it has been shown to be a rate-limiting step in fat oxidation (51). It should be noted, however, that despite the apparent increase in muscle energy production from fat in response to prednisone administration, there was no change in the whole-body substrate use during the study as measured by indirect calorimetry. It is possible that shifts in fuel metabolism were localized only to muscle or that the muscle adaptations preceded other steps in fat mobilization and transport required to actually alter substrate use. These possibilities require further study.

In conclusion, the current study demonstrates that short-term use of a moderate dose of prednisone has no effect on whole-body or leg muscle protein metabolism. There is also no effect on muscle strength or muscle mitochondrial function. Circulating glucose and insulin levels are elevated in response to prednisone, indicating that glucose metabolism is more affected than protein metabolism by glucocorticoids.

	Acknowledgments

 
We thank Jane Kahl, Rebecca Kurup, Dawn Morse, and Jill Schimke for their technical assistance with sample analysis and Chan Boyer, Charles Ford, Jaime Gransee, and Mai Persson for mass spectrometric analysis. We also thank the Department of Radiology and members of the GCRC dietary, nursing, and support staff for their help in carrying out these studies.

	Footnotes

 
This work was supported by National Institutes of Health Grants RO1-DK41973 (to K.S.N.), T32-DK07352 (to K.R.S.), and MO1-RR00585. Additional support was provided by the Mayo Foundation and the Murdock-Dole Professorship (to K.S.N.) and the Mayo-Thompson Fellowship (to K.R.S.). J.N. was supported by the Swedish Society of Medicine, The Medical Research Council (09101), the Henning and Johan Throne-Holsts Foundation, and the Wenner-Gren Center Foundation.

Abbreviations: COX, Cytochrome c oxidase; CS, citrate synthase; GC/MS, gas chromatograph/mass spectrometer; HAD, L-3-hydroxyacyl coenzyme A dehydrogenase; IGFBP, IGF binding protein; KIC, ketoisocaproate; Leu, leucine; Phe, phenylalanine; Qpt, Phe conversion to Tyr; Ra, rate of appearance; Rd, rate of disappearance; Tyr, tyrosine.

Received May 13, 2004.

Accepted September 14, 2004.

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