Effect of Short-Term Prednisone Use on Blood Flow, Muscle Protein Metabolism, and Function
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0021-972X/04/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 89(12):6198 – 6207
Printed in U.S.A. Copyright © 2004 by The Endocrine Society
doi: 10.1210/jc.2004-0908
Effect of Short-Term Prednisone Use on Blood Flow,
Muscle Protein Metabolism, and Function
KEVIN R. SHORT, JONAS NYGREN, MAUREEN L. BIGELOW, AND K. SREEKUMARAN NAIR
Endocrinology Research Unit, Mayo Clinic School of Medicine, Rochester, Minnesota 55905
Glucocorticoids can cause muscle atrophy, but the effect on blood flow was 25% lower on Pred leading to 15–30% lower
muscle protein metabolism in humans has not been ade- amino acid flux among the artery, vein, and muscle. However,
quately studied to know whether protein synthesis, break- amino acid net balance and rates of protein synthesis and
down, or both are altered. We tested the effect of 6 d of oral breakdown were unchanged, as were synthesis rates of total
prednisone (Pred, 0.5 mg/kg䡠d) on muscle protein metabolism mixed, mitochondrial, sarcoplasmic, and myosin heavy chain
and function. Six healthy subjects (three men/three women, muscle proteins. Muscle mitochondrial function, muscle
22– 41 yr) completed two trials (randomized, double-blind, strength, and resting energy expenditure were also un-
cross-over) with Pred and placebo. Fasting glucose, insulin, changed. These results demonstrate that a short-term mod-
IGF-I, and glucagon were higher on Pred vs. placebo, whereas erate dose of prednisone affects glucose metabolism but has
IGF-II and IGF binding protein-1 and -2 were lower. Whole- no effect on whole-body or leg muscle protein metabolism or
body amino acid fluxes, blood urea nitrogen, and urinary ni- muscle function. (J Clin Endocrinol Metab 89: 6198 – 6207,
trogen loss were not statistically different between trials. Leg 2004)
G LUCOCORTICOIDS ARE EXTENSIVELY used to treat
clinical conditions such as inflammation, asthma, and
immune suppression. However, there are undesirable effects
limited to measurements of whole-body amino acid kinetics
in young healthy people after short-term glucocorticoid ad-
ministration. It was observed in some of those studies that
that arise in the presence of excess glucocorticoids, including protein breakdown, as assessed by the appearance rate of
inhibition of insulin action on glucose metabolism (1– 4) and leucine (Leu) or phenylalanine (Phe) using amino acid trac-
muscle wasting and weakness (5–7). A reduction in muscle ers, is increased after 6 –7 d of moderate- to high-dose (0.5–
mass implies that glucocorticoids alter the balance between 0.8 mg/kg䡠d) prednisone administration in healthy volun-
protein synthesis and breakdown, although the exact mech- teers (19 –22). Whole-body oxidation of Leu may also be
anisms have not been fully clarified. increased by glucocorticoids (19 –22), whereas whole-body
Studies in rodents have shown that high-dose glucocor- protein synthesis is typically unchanged (19, 20, 22) or
ticoid administration increases protein breakdown in skel- slightly decreased (21). However, because whole-body stud-
etal muscle by enhancing the expression and activity of com- ies represent the average protein turnover of all of the body
ponents of protein catabolism pathways (8 –11). There is also protein pools, it is not possible to determine whether the
evidence that muscle protein synthesis is suppressed and results reflect changes in individual tissues, such as skeletal
that this is due, at least in part, to inhibition of the complexes muscle.
involved in initiation of protein translation (12–16). Whereas There are limited data available on the effect of glucocor-
these studies have been useful for identifying the potential ticoids on human muscle protein metabolism. Beaufrere et al.
events that lead to muscle wasting, their applicability to (19) measured the appearance of urinary 3-methyl histidine,
humans is limited by the fact that the doses of glucocorticoids an index of muscle protein breakdown, and observed no
given experimentally to rats are much higher than would change between prednisone (0.8 mg/kg䡠d for 5 d) and pla-
typically be administered to humans. Furthermore, some of cebo trials. This led the authors to conclude muscle protein
the effects attributed to glucocorticoids, such as the dramatic breakdown was not affected by short-term glucocorticoid
loss of body and muscle weight, may be due in part to the elevation. However, because nonmuscle tissues, such as the
anorexic effect that occurs in rodents in response to these gut, can produce 3-methyl histidine, this measure may not be
high doses (12, 17, 18). sufficiently specific and sensitive to detect the treatment ef-
Nearly all of the previous human studies examining the fect in muscle. This observation also does not rule out the
effect of glucocorticoids on protein metabolism have been possibility that glucocorticoids may have an effect on muscle
protein synthesis rate. In two other studies, arteriovenous
Abbreviations: COX, Cytochrome c oxidase; CS, citrate synthase; amino acid balance across the forearm was examined in
GC/MS, gas chromatograph/mass spectrometer; HAD, l-3-hydroxya- young healthy people after 4 d of oral dexamethasone (8
cyl coenzyme A dehydrogenase; IGFBP, IGF binding protein; KIC, ke- mg/d), which has higher potency than prednisone (3, 4). In
toisocaproate; Leu, leucine; Phe, phenylalanine; Qpt, Phe conversion to
Tyr; Ra, rate of appearance; Rd, rate of disappearance; Tyr, tyrosine. those studies, the authors pointed to a nonstatistically sig-
JCEM is published monthly by The Endocrine Society (http://www.
nificant trend for more negative net balance of Phe in the
endo-society.org), the foremost professional society serving the en- fasted state as evidence that glucocorticoid use resulted in
docrine community. greater tissue protein loss. However, neither amino acid up-
6198
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 November 2015. at 21:40 For personal use only. No other uses without permission. . All rights reserved.Short et al. • Prednisone Effect on Skeletal Muscle J Clin Endocrinol Metab, December 2004, 89(12):6198 – 6207 6199
take (a marker of protein synthesis) nor appearance (from electrolytes, and glucose), complete blood count, urinalysis, and elec-
protein breakdown) across the forearm was significantly al- trocardiogram. Inclusion criteria included age (18 – 45 yr) and body mass
index (20 –28 kg/m2). Exclusion criteria included tobacco use, -blockers
tered by glucocorticoid treatment (3, 4). Only one study (23) or any medications that could affect metabolism or blood coagulation,
has directly measured muscle protein synthesis rate using diabetes or other endocrine disorders, and debilitating chronic illness.
muscle biopsy methods in humans. In that study, women None of the participants were taking medications at the time of the
(mean age 58 –71 yr) with rheumatoid arthritis undergoing study, nor were they engaged in a regular exercise program.
knee surgery who had used prednisone for 9 yr had a 30%
lower rate of synthesis of mixed (total) muscle proteins in the Protocol and procedures
quadriceps muscle, compared with arthritis patients who
Each participant completed two similar trials separated by an average
had not used corticosteroids (23). It is unclear, however, how
of 6 wk (range 5– 8 wk). Regular lifestyle patterns were maintained
much the disease status, physical activity history, and sur- between trials. During each study period, either prednisone or placebo
gical treatment of these patients contributed to the observed was administered in a randomized, double-blind manner for 6 d. Cap-
effects. This finding requires confirmation under well-con- sules containing prednisone (0.5 mg/kg䡠d) or placebo were consumed
trolled conditions. Thus, at the present time, there are no 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
studies that have simultaneously used arteriovenous balance Pharmacy Department and were indistinguishable from each other.
and muscle biopsy methods to comprehensively determine During each study period, a weight-maintaining diet (55:30:15% carbo-
whether excess glucocorticoids alter protein synthesis hydrate, fat, and protein, respectively) was provided on d 3–5 of the
and/or breakdown in human skeletal muscle. treatment. Strenuous physical activity was avoided on d 3–5. On the
morning of d 5, muscle strength testing was performed, as described
In the current investigation, we tested whether short-term
below. That evening (1800 h), participants were admitted to the General
(6 d) administration of glucocorticoids would alter muscle Clinical Research Center (GCRC) for in-patient study. A light snack was
protein metabolism in healthy subjects. We used these ex- provided at 2200 h, and no food was consumed thereafter until com-
perimental conditions to avoid confounding factors that pletion of the study the next day.
might arise if the studies were performed in patients with The following morning (d 6), the last dose of prednisone or placebo
was taken at approximately 0530 h. Within the next hour, a polyethylene
disease and so that our data would be comparable with venous catheter was placed in an antecubital arm vein for infusion of
previous human studies. To perform a more comprehensive isotopic tracers. Primed, continuous infusions of [1,2-13C]Leu (10.4
evaluation of protein metabolism than prior work in this mol/kg prime, 10.4 mol/kg䡠h thereafter), [15N]Phe (4.2 mol/kg
area, amino acid kinetics were measured at the level of the prime, 4.2 mol/kg䡠h thereafter), [2H4]Tyr (3.0 mol/kg prime, 3.0
whole body and across the leg, using arteriovenous balance mol/kg䡠h thereafter), and [15N]Tyr (1.6 mol/kg prime only) were
maintained for 8 h. The start of the infusion is designated as time 0 min.
techniques. Muscle biopsies of the vastus lateralis were also Once the infusion of tracers was started, subjects were transported a
obtained to measure the fractional synthesis rates of muscle short distance to the Vascular Radiology Laboratory for placement of
proteins and the oxidative capacity of the tissue. Results were lines in the femoral artery and vein for infusion and sample collection
confirmed using multiple amino acid tracers. (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
Subjects and Methods the catheter tip in the common iliac artery. This catheter was used for
arterial blood sampling, and the sheath was used to infuse indocyanine
Materials green. The distal tip of the venous sheath was placed in the external iliac
l-[1,2-13C]Leu (97 atom percent excess) was purchased from Mass vein a few centimeters above the inguinal ligament. The volunteers were
Trace (Woburn, MA) and Isotec Inc. (Miamisburg, OH). l-[15N]Phe (97 then transferred back to the GCRC for completion of the study. The
atom percent excess), l-[15N]tyrosine (Tyr, 97 atom percent excess), and arterial and venous lines were maintained by normal saline infusion.
[2H4]Tyr (91 atom percent excess) were purchased from Cambridge Leg blood flow was determined by indicator-dilution technique dur-
Isotope Laboratories, Inc. (Woburn, MA). Isotopes were tested before ing arterial infusion of indocyanine green from 120 to 210 min and again
use for their isotopic and chemical purity. The isotope solutions were from 390 to 480 min (24, 25). Blood samples were drawn from the femoral
prepared under sterile conditions and were determined to be bacteria artery and vein at 150, 170, 190, and 210 min and again at 420, 440, 460,
and pyrogen free before their administration to humans. Luciferin/ and 480 min. Muscle biopsies of the vastus lateralis were obtained under
luciferase reagent for ATP monitoring (formula SL) was purchased from local anesthesia at 240 and 480 min of tracer infusion (26). The biopsies
BioThema (Haninge, Sweden) and ADP and ATP from Roche Molecular were performed on the same leg that was catheterized, and the second
Biochemicals (Indianapolis, IN). All other reagents for mitochondrial biopsy site was approximately 8 –10 cm proximal from the first biopsy.
assays were purchased from Sigma Chemicals (St. Louis, MO). The study A portion of the muscle was kept on ice in saline-soaked gauze for
protocol was approved by the Institutional Review Board of Mayo mitochondrial studies, as described below. The remainder of the tissue
Foundation. All procedures were performed in accordance with the was rapidly frozen in liquid nitrogen and stored at ⫺80 C. Resting
ethical guidelines of the Declaration of Helsinki and were clearly ex- energy expenditure was determined by indirect calorimetry (DeltaTrac,
plained to the study volunteers during their initial visit. Each participant SensorMedics, Yorba Linda, CA) for 45 min beginning at approximately
provided his or her informed oral and written consent before enrollment 270 min. The last 30 min of this measurement were used for data analysis.
into the study. Upon completion of the study, leg catheters were removed and partic-
ipants remained overnight for observation before being discharged from
the GCRC.
Participants
Six young, healthy people (three men, three women) volunteered to Hormone and metabolite assays
participate in the study after responding to advertisements placed in the
local area (Rochester, MN). Average characteristics (mean ⫾ sem) of the Glucose was measured with a Beckman glucose analyzer (Beckman
group were: age 30 ⫾ 3 yr, height 173 ⫾ 2 cm, weight 72.6 ⫾ 3.5 kg, body Instruments, Porterville, CA). Nonesterified free fatty acids were mea-
mass index 24.2 ⫾ 1.0 kg/m2, body fat-free mass 51.1 ⫾ 3.9 kg, and body sured using an enzymatic colorimetric assay (NEFA C; Wako Chemicals
fat 24.4 ⫾ 3.4%. Body composition was determined using dual-energy USA, Richmond, VA). Plasma levels of amino acids were measured by
x-ray absorptiometry. Health status was assessed by medical history, an HPLC system (HP 1090, 1046 fluorescence detector and cooling sys-
physical exam, blood chemistries (including liver enzymes, creatinine, tem) with precolumn O-phthalaldehyde derivatization (27). Urinary
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nitrogen content was measured using a Beckman GM7 Analox Microstat Hercules, CA) and purified the next day using a column of the same
(Beckman Instruments). resin. The amino acids were dried (SpeedVac, Savant Instruments,
Insulin and human GH were measured with two-site immunoenzy- Hicksville, NY) and then derivitized as their trimethyl acetyl methyl
matic assays (Access system, Beckman Instruments, Chaska, MN). Glu- ester. [13C]Leu and [15N]Phe enrichments in muscle proteins were de-
cagon was measured by a direct, double-antibody RIA (Linco Research, termined using a gas chromatograph-combustion-isotope ratio mass
St. Louis, MO). spectrometer (Delta Plus, Finigan MAT) as described previously (35, 36).
After separation from their binding proteins with a simple organic Tissue fluid amino acids and amino-acyl tRNA samples were derivat-
solvent, total IGF-I and IGF-II were measured with two-site immuno- ized as their t-butyldimethylsilyl ester and analyzed for [13C]Leu and
radiometric assays (Diagnostic Systems Laboratories, Webster, TX). IGF [15N]Phe enrichments using a GC/MS (HP5973, Hewlett-Packard In-
binding protein (IGFBP)-1 and -3 were also measured with two-site struments) under electron ionization conditions (34, 35, 37).
immunoradiometric assays, whereas IGFBP-2 was measured by a The fractional synthetic rates of mitochondrial and sarcoplasmic pro-
double-antibody RIA (Diagnostic Systems Laboratories). teins were calculated using the equation,
fractional synthetic rate (percent per hour)
Plasma amino acid kinetics
⫽ 100 ⫻ 共E8 h ⫺ E3 h兲/共Ep ⫻ T兲,
The enrichment level of [1,2-13C]Leu in plasma was determined using
a gas chromatograph/mass spectrometer (GC/MS; HP5973, Hewlett- where (E8 h ⫺ E3 h) represents the increment in [13C]Leu or [15N]Phe
Packard Instruments, Avondale, CA) by multiple ion monitoring at m/z enrichment in muscle proteins between 3 and 8 h of infusion. Ep is the
342/344 under positive ion methane chemical ionization conditions. The average precursor pool enrichment of [13C]Leu or [15N]Phe in either
concentration of l-Leu was simultaneously determined by comparison muscle tissue fluid or amino-acyl tRNA taken from the 3- and 8-h
with a norleucine internal standard. [15N]Phe, [15N]Tyr, and [2H4]Tyr biopsies. T is the time of incorporation between the two biopsies, which
were measured as their t-butyldimethylsilyl ester derivatives under in this case was 5 h.
electron ionization conditions using a gas chromatograph/mass spec-
trometer (Voyager, Finigan MAT, Bremen, Germany). Fragment ions
were monitored at m/z 345/337/336 for Phe and m/z 472/470/467/466 Muscle oxidative capacity
for Tyr. [1,2-13C]Ketoisocaproate (KIC) in plasma was determined as its Mitochondria were isolated by centrifugation from fresh muscle tis-
quinoxalinol-trimethylsilyl ether derivative under electron ionization sue, and ATP production capacity was assessed using a bioluminescent
conditions using an HP5988 GC/MS (Hewlett-Packard) (28). KIC con- method as previously described (38, 39). Briefly, mitochondria were
centration was measured simultaneously in the same samples by com- added to cuvettes containing luciferin, luciferase, 0.3 mm ADP, and one
parison with ketoisovalerate, which was added as an internal standard. of six substrate combinations. Substrates used were, in mm, 10 glutamate
All samples were analyzed in duplicate. ⫹ 1 malate, 10 ␣-ketoglutarate, 1 pyruvate ⫹ 1 malate, 0.05 palmitoyl-
For calculation of whole-body amino acid kinetics, the mean values l-carnitine ⫹ 1 malate, 20 succinate ⫹ 0.1 rotenone, or 1 pyruvate ⫹ 0.05
of isotopic enrichment from 3 to 8 h of infusion were used. Whole-body palmitoyl-l-carnitine ⫹ 10 ␣-ketoglutarate ⫹ malate. ATP production
flux rates of Leu, Phe, and Tyr were calculated by tracer dilution using was measured simultaneously for all reactions in triplicate at 25 C in
the equation, Q ⫽ i[(Ei/Ep) ⫺ 1], where Q represents flux of a particular BioOrbit 1251 luminometer (BioOrbit Oy, Turku, Finland). Each reaction
amino acid, i is the rate of tracer infusion, and Ei and Ep are the was calibrated using an internal ATP standard. A separate piece of
enrichment of the tracer in the infusate and the plasma at isotopic muscle (20 mg) was homogenized as a buffer containing 20 mm HEPES,
plateau, respectively. For Tyr flux the enrichment of [2H4]Tyr was used. 1 mm EDTA, and 250 mm sucrose (pH 7.4), supplemented with a pro-
The Phe conversion to Tyr (Qpt) was calculated as previously reported tease inhibitor cocktail (Complete Mini, Roche Applied Science, India-
(29, 30). The Phe incorporation into protein (Sp) for whole body is napolis, IN). Aliquots of the homogenate were used to measure protein
calculated by subtracting Qpt from Qp because Phe is either irreversibly concentration (DC protein assay, Bio-Rad Laboratories) and the activity
converted into Tyr or incorporated into protein (29, 30). of the mitochondrial enzymes citrate synthase (CS, from the Krebs cycle),
Calculation of amino acid kinetics across the leg was performed using cytochrome c oxidase (COX, part of the respiratory chain), and l-3-
two methods. The first method used arterial and venous amino acid hydroxyacyl coenzyme A dehydrogenase (HAD, a step in fatty acid
concentration and enrichment and a measure of blood flow (24, 25). This -oxidation) using spectrophotometric assays at 25 C (32, 39, 40).
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 Muscle strength tests
Rd is a measure of amino acids leaving the circulation into tissue. The Three tests of upper-body strength were conducted on the morning
second calculation was a three-pool model that also used arterial and of d 5 of each study phase. Isometric handgrip strength was determined
venous amino acid concentration and enrichment and blood flow as well from a series of six maximal efforts. The best of the six trials was taken
as a measure of the intracellular enrichment of the tracer in the tissue of for data analysis. Chest press and arm (biceps) curl strength were mea-
interest, which in this case was skeletal muscle (31). Although the model sured as the one-repetition maximum weight lifted during a progressive
was originally developed for use with muscle tissue fluid (free amino series of attempts. Two familiarization sessions were completed ap-
acid pool), it has been recently demonstrated that the derived flux values proximately 1 and 2 wk before commencing the study. This assured that
are significantly different from flux values calculated using enrichment the subjects could reliably generate maximal efforts with a minimal
in the amino-acyl tRNA pool (31). The advantage of using amino-acyl number of attempts. No lower-body exercises were performed to min-
tRNA for this purpose is that it is assumed to reflect the tracer enrich- imize the chance that muscle activation would affect protein metabolism
ment in the immediate precursor pool for protein synthesis, whereas the of the legs on the following day.
tissue fluid pool is a mixture of both intracellular (⬃85%) and extracel-
lular (⬃15%) free amino acids. Therefore in the current study, we used
amino-acyl tRNA for these calculations.
Statistical analysis
Summarized values are reported as mean ⫾ sem. Paired t tests were
Muscle protein synthesis used for comparisons between placebo and prednisone trials, with
␣-level set to 5% to define statistical significance.
A 150-mg portion of each muscle sample was used for the isolation
of mitochondrial and sarcoplasmic protein fractions by differential cen- Results
trifugation as previously described (26, 32, 33). A separate 20- to 30-mg
piece of muscle was used to prepare total mixed muscle proteins and Plasma metabolites and hormones
isolate free tissue fluid amino acids (34). Amino-acyl tRNA was isolated
from a 150-mg piece of muscle (34). Compared with the placebo condition, prednisone re-
The muscle protein fractions were hydrolyzed overnight in 0.6 m HCl sulted in statistically increased levels of circulating glucose,
in the presence of cation exchange resin (AG-50, Bio-Rad Laboratories, insulin, C-peptide, glucagon, and IGF-I (Table 1). There was
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TABLE 1. Plasma metabolites and hormones
Placebo Prednisone % Difference P value
Glucose [mg/dl (mmol/liter)] 88 ⫾ 2 102 ⫾ 5 16 0.003
(4.8 ⫾ 0.1) (5.7 ⫾ 0.3)
NEFA (mmol/liter) 0.778 ⫾ 0.067 0.949 ⫾ 0.075 22 0.068
C-peptide [ng/ml (nmol/liter)] (0.97 ⫾ 0.11) (1.57 ⫾ 0.18) 63 0.001
(0.32 ⫾ 0.04) (0.52 ⫾ 0.06)
Glucagon [pg/ml (ng/liter)] 85.8 ⫾ 22.4 96.2 ⫾ 22.1 12 0.001
(85.8 ⫾ 22.4) (96.2 ⫾ 22.1)
GH [ng/ml (g/liter)] 0.86 ⫾ 0.25 0.88 ⫾ 0.31 2 0.967
(0.86 ⫾ 0.25) (0.88 ⫾ 0.31)
IGF-I (total ng/ml) 281 ⫾ 45 379 ⫾ 49 35 0.042
IGF-II (ng/ml) 482 ⫾ 54 451 ⫾ 51 ⫺6 0.027
IGFBP-1 (ng/ml) 40.01 ⫾ 7.95 22.22 ⫾ 9.19 ⫺44 0.010
IGFBP-2 (ng/ml) 318 ⫾ 95 276 ⫾ 100 ⫺13 0.020
IGFBP-3 (ng/ml) 3699 ⫾ 289 3751 ⫾ 245 1 0.685
Insulin [U/ml (pmol/liter)] 3.97 ⫾ 0.92 8.28 ⫾ 1.64 109 0.004
(24 ⫾ 6) (50 ⫾ 10)
also a trend for increased nonesterified fatty acids (P ⫽ 0.068) TABLE 2. Amino acid and KIC concentrations in arterial plasma
during the prednisone trial. During the prednisone trial, (mean ⫾ SEM)
levels of IGF-II, IGFBP-1, and IGFBP-2 were significantly Amino acid Placebo Prednisone % Difference P value
reduced, whereas GH and IGFBP-3 levels were not statisti-
Alanine 1.31 ⫾ 0.16 1.68 ⫾ 0.23 28 0.117
cally different from the placebo trial. There were no statistical (147 ⫾ 18) (189 ⫾ 26)
differences between trials in the concentration of any plasma Arginine 1.69 ⫾ 0.11 1.43 ⫾ 0.13 ⫺15 0.104
amino acids or ␣KIC (Table 2). (97 ⫾ 7) (82 ⫾ 7)
Glutamate 0.68 ⫾ 0.06 0.67 ⫾ 0.06 ⫺2 0.636
(46 ⫾ 4) (45 ⫾ 4)
Energy expenditure measured by indirect calorimetry
Glutamine 6.51 ⫾ 0.88 6.18 ⫾ 0.55 ⫺5 0.560
In the overnight fasting state, the respiratory exchange (445 ⫾ 60) (423 ⫾ 38)
ratio was 0.76 ⫾ 0.02 during the placebo trial and 0.75 ⫾ 0.02 Glycine 1.27 ⫾ 0.12 1.17 ⫾ 0.12 ⫺8 0.208
(170 ⫾ 19) (156 ⫾ 17)
during the prednisone trial (P ⫽ 0.908), indicating that sub- Histidine 1.24 ⫾ 0.12 1.04 ⫾ 0.11 ⫺16 0.095
strate use between trials was similar. Likewise, there were no (80 ⫾ 8) (67 ⫾ 7)
significant differences between trials in resting oxygen con- Isoleucine 0.65 ⫾ 0.07 0.57 ⫾ 0.08 ⫺12 0.241
sumption (257 ⫾ 16 vs. 281 ⫾ 22 ml/min for placebo and (49 ⫾ 6) (43 ⫾ 6)
Ketoisocaproate 0.45 ⫾ 0.03 0.47 ⫾ 0.03 4 0.243
prednisone, respectively, P ⫽ 0.168) or resting metabolic rate (34.9 ⫾ 2.0) (36.4 ⫾ 2.2)
(73 ⫾ 5 vs. 80 ⫾ 6 kcal/h for placebo and prednisone, re- Leucine 1.80 ⫾ 0.19 1.51 ⫾ 0.17 ⫺16 0.206
spectively, P ⫽ 0.184). (137 ⫾ 15) (115 ⫾ 13)
Lysine 2.12 ⫾ 0.07 1.91 ⫾ 0.21 ⫺10 0.365
Protein kinetics and leg blood flow (145 ⫾ 5) (130 ⫾ 15)
Methionine 0.50 ⫾ 0.03 0.47 ⫾ 0.06 ⫺5 0.467
Blood urea nitrogen levels were not statistically different (33 ⫾ 2) (32 ⫾ 4)
between trials [13 ⫾ 2 mg/dl (4.5 ⫾ 0.6 mmol/liter) for Phenylalanine 0.81 ⫾ 0.05 0.73 ⫾ 0.08 ⫺10 0.283
(49 ⫾ 3) (44 ⫾ 5)
placebo and14 ⫾ 2 mg/dl (4.9 ⫾ 0.8 mmol/liter) for pred- Serine 0.90 ⫾ 0.12 0.77 ⫾ 0.06 ⫺15 0.202
nisone, P ⫽ 0.493]. Urinary nitrogen loss during the study (86 ⫾ 11) (73 ⫾ 6)
day tended to be higher during the prednisone trial [0.22 ⫾ Threonine 1.26 ⫾ 0.14 1.18 ⫾ 0.15 ⫺6 0.565
0.04 g/h (16 ⫾ 3 mmol/h)] than during placebo [0.17 ⫾ 0.03 (106 ⫾ 12) (99.4 ⫾ 13.0)
g/h (12 ⫾ 5 mmol/h)], but the difference was not statistically Tyrosine 1.00 ⫾ 0.09 0.90 ⫾ 0.08 ⫺10 0.453
(55.0 ⫾ 4.9) (50 ⫾ 4)
significant (P ⫽ 0.111). Leg blood flow while on prednisone Valine 1.82 ⫾ 0.27 1.48 ⫾ 0.17 ⫺18 0.186
(33 ⫾ 6 ml/min䡠kg leg fat-free mass) was 25% lower (P ⫽ (155 ⫾ 23) (127 ⫾ 14)
0.060) than during the placebo trial (45 ⫾ 5 ml/min䡠kg leg Primary values are shown in micrograms per deciliter; values in
fat-free mass). Enrichment of the free amino acid pools in parentheses are shown in micromoles per liter.
plasma and muscle as well as the amino-acyl tRNA and
protein-bound enrichments in muscle are shown in Table 3. inward and outward flux as well as the fluxes from artery to
Whole-body amino acid fluxes of Leu and Phe, shown in vein (also known as shunting), tissue to vein, and artery to
Fig. 1, were not statistically different between the study treat- tissue were 16 –36% lower in the prednisone trial vs. placebo.
ments. Likewise, the Ra, Rd, and net balance of Leu, Phe, and These differences reached statistical significance or showed
Tyr across the leg were not significantly affected by the a strong trend (P ⬍ 0.12) for both tracers. However, protein
treatment conditions (Fig. 2). Flux rates of Phe and Leu cal- breakdown and synthesis rates calculated with the three-
culated from the three-pool model (31) are shown in Fig. 3. pool model using either of the tracers were not significantly
The requirement that tracer enrichment in the three com- altered by prednisone use (both P ⬎ 0.50).
partments follow the pattern [artery ⬎ vein ⬎ tissue (amino- Figure 4 shows the fractional synthesis rate of total mixed
acyl tRNA is used for tissue)] was met for all cases. The muscle proteins and the mitochondrial and sarcoplasmic
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TABLE 3. Tracer enrichments in plasma and muscle pools (mean ⫾ SEM)
Placebo Prednisone % Difference P value
Plasma free pools
1,2-[13C2]Leu 9.93 ⫾ 0.16 9.90 ⫾ 0.29 0 0.919
1,2-[13C2]KIC 8.51 ⫾ 0.17 8.36 ⫾ 0.27 ⫺2 0.326
[15N]Phe 10.29 ⫾ 0.26 9.97 ⫾ 0.18 ⫺3 0.245
[15N]Tyr 1.75 ⫾ 0.08 1.57 ⫾ 0.05 ⫺10 0.142
[2H4]Tyr 9.60 ⫾ 0.69 8.44 ⫾ 0.78 ⫺12 0.196
Muscle pools
Tissue fluid [13C2]Leu 7.25 ⫾ 0.25 6.76 ⫾ 0.29 ⫺7 0.059
Tissue fluid [15N]Phe 7.23 ⫾ 0.31 6.56 ⫾ 0.30 ⫺9 0.026
[13C2]Leu-tRNA 5.00 ⫾ 0.42 4.56 ⫾ 0.43 ⫺9 0.437
[15N]Phe-tRNA 3.87 ⫾ 0.36 3.17 ⫾ 0.28 ⫺18 0.048
Muscle protein-bound pools
Mixed protein [13C2]Leu 0.026 ⫾ 0.002 0.026 ⫾ 0.002 2 0.872
Mixed protein [15N]Phe 0.021 ⫾ 0.002 0.020 ⫾ 0.002 ⫺5 0.685
Mitochondrial protein [13C2]Leu 0.027 ⫾ 0.006 0.029 ⫾ 0.003 10 0.461
Mitochondrial protein [15N]Phe 0.023 ⫾ 0.004 0.024 ⫾ 0.002 3 0.280
Sarcoplasmic protein [13C2]Leu 0.019 ⫾ 0.001 0.023 ⫾ 0.003 17 0.382
Sarcoplasmic protein [15N]Phe 0.016 ⫾ 0.001 0.020 ⫾ 0.003 28 0.217
Plasma and muscle precursor values are expressed as mole percent excess; muscle protein-bound values in atom percent excess.
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.
subfractions calculated using either tissue fluid or amino-
acyl tRNA as precursor pool. There were no statistically
significant differences in synthesis rates of muscle proteins as
calculated from either the Leu or Phe tracer data.
Muscle function
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 FIG. 2. Amino acid kinetics across the leg using standard dilution
equations (24, 25). Net balance of Leu, Phe, and Tyr were all negative,
all but one substrate combination. The exception was that indicating a net release of amino acids during the postabsorptive state
there was a small (12%) but statistically significant increase (top). The Ra and Rd for Leu, Phe, and Tyr are shown in the bottom
in ATP production with palmitoyl-l-carnitine and malate panel. Paired t test comparisons between treatments all had P ⬎ 0.45.
during the prednisone trial vs. placebo. FFM, Fat-free mass.
Muscle strength was also not statistically different be-
tween treatments. The peak values for placebo and pred- adversely affected by 6 d of oral prednisone administration
nisone conditions, respectively, were 67.2 ⫾ 10.4 and 65.9 ⫾ at a dose of 0.5 mg/kg䡠d in healthy people. Muscle strength
11.1 kg for chest press, 44.6 ⫾ 6.9 and 43.6 ⫾ 7.4 for arm curl, and all but one index of muscle oxidative capacity were also
and 47.0 ⫾ 3.6 and 46.3 ⫾ 3.4 kg for isometric handgrip (all unaffected. In contrast, postabsorptive levels of plasma glu-
P ⬎ 0.15). cose and several hormones were significantly altered and leg
blood flow was reduced. This suggests that carbohydrate
Discussion
metabolism and other endocrine systems are relatively more
The results of the current study demonstrate that postab- sensitive to the effects of glucocorticoids than whole-body
sorptive whole-body and muscle protein metabolism is not and muscle protein metabolism.
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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. Num-
bers over bars are P values for paired t test
comparisons between treatments. FFM, Fat-
free mass.
FIG. 4. Synthesis rates of muscle proteins.
Rates are shown for total mixed muscle pro-
teins and the mitochondrial (Mito) and sarco-
plasmic (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 be-
tween treatments all had P values ⬎ 0.20.
We hypothesized that prednisone use would result in artery, femoral vein, and leg muscle tissue as calculated by
higher muscle protein breakdown and/or reduced muscle the three-pool model (Fig. 3). To our knowledge, the present
protein synthesis. There was a strong trend for leg blood flow study is the first to report a reduction in leg blood flow in
to be lower during the prednisone trial, which may account humans in response to glucocorticoids. The reduction in leg
for the modest changes in flux rates among the femoral blood flow occurred in five of the six participants (the sixth
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TABLE 4. Activity of oxidative enzymes and mitochondrial ATP production capacity in skeletal muscle (mean ⫾ SEM)
Placebo Prednisone % Difference P value
Citrate synthase activity 24.26 ⫾ 2.38 24.67 ⫾ 2.21 2 0.588
COX activity 9.47 ⫾ 1.12 8.58 ⫾ 1.34 ⫺9 0.415
HAD activity 9.34 ⫾ 0.92 9.96 ⫾ 0.54 7 0.360
Mitochondrial ATP production rate
Glutamate ⫹ malate 9.41 ⫾ 0.88 9.68 ⫾ 0.78 3 0.380
PPKM 8.29 ⫾ 0.91 8.52 ⫾ 0.73 3 0.561
Ketoglutarate 6.59 ⫾ 0.70 6.77 ⫾ 0.69 3 0.576
Pyruvate ⫹ malate 4.28 ⫾ 0.44 4.02 ⫾ 0.47 ⫺6 0.203
Palmitoyl-L-carnitine ⫹ malate 3.21 ⫾ 0.39 3.60 ⫾ 0.45 12 0.004
Succinate ⫹ Rotenone 3.99 ⫾ 0.66 3.85 ⫾ 0.53 ⫺4 0.613
Mitochondrial ATP production rates are shown for six different substrate combinations. PPKM, Pyruvate ⫹ palmitoyl-L-carnitine ⫹
␣-ketoglutarate ⫹ malate. All values are expressed in micromoles per minute per gram of muscle.
had no change). In two previous studies conducted after 4 d short-term glucocorticoid use (3, 19 –22). In those studies
of dexamethasone administration, there were no statistically whole-body Leu oxidation was also increased and whole-
significant changes in forearm blood flow detected in the body protein synthesis was either unchanged or decreased
basal state (3, 4), although there was a trend for a 20% re- (19 –22). Thus, the balance of amino acid metabolism was
duction in flow during the dexamethasone trial in one of shifted in favor of a more catabolic state by glucocorticoids,
those reports (3). Recent work in pigs revealed that a single and this was supported by increased loss of urinary nitrogen
pharmacological dose of prednisone results in reductions in (3, 21, 22). Surprisingly, however, it was reported that whole-
blood flow to the muscle, skin, and bone in the hip area that body amino acid kinetics were not altered in patients with
are evident within 1 h and persist at least 24 h (41). This rapid Cushing’s syndrome (43). This latter finding requires con-
onset of effect suggests that the blood flow reduction arises firmation in specific studies examining skeletal muscle me-
from so-called nongenomic effects of glucocorticoids that are tabolism because it is inconsistent with the loss of protein
not mediated through transcription or translation. A poten- mass in these patients. Unlike those earlier reports, we did
tial mechanism for the effect on blood flow was revealed by not detect a significant alteration in whole-body protein
a recent study that showed that glucocorticoids have a det- breakdown after prednisone administration. The reason for
rimental effect on vascular epithelial cells (42). In that study this discrepancy as well as the lack of prednisone effects on
human umbilical vein epithelial cells exposed to dexameth- muscle protein turnover is not yet clear. However, the
asone produced less nitric oxide, apparently due to higher strength of the current investigation was that the study out-
presence of oxidants such as hydrogen peroxide and per- comes were confirmed with multiple amino acid tracers at
oxynitrite. Thus, those authors proposed that reduced nitric the whole-body, arteriovenous, and muscle protein levels
oxide production could prevent vasorelaxation leading to using some of the most detailed techniques currently avail-
reduced blood flow as well as higher risk of vascular com- able. We used both a compartmental analysis model recently
plications for long-term glucocorticoid users (42). developed to measure leg muscle protein kinetics (31) as well
Despite the lower blood flow that led to reduced amino as direct measurement of the fractional synthesis rate of
acid movement through the leg, there was no change in muscle proteins using amino acyl t-RNA as the precursor
protein breakdown or synthesis detected using either the Leu pool. Blood urea nitrogen and urinary nitrogen losses were
or Phe tracer or with the different methods of calculation. also not significantly altered. Thus, several independent
There was also no change in the fraction synthesis rate of measurements corroborate the lack of effect of prednisone on
mixed (total) muscle proteins, or the subfractions of mito- whole-body and muscle protein kinetics in this study.
chondrial, sarcoplasmic, or myosin heavy-chain proteins. The dose and duration of prednisone administered in the
The effect of glucocorticoid administration on fractional syn- current study (0.5 mg/kg䡠d for 6 d) was at the lower range
thesis rate of muscle proteins humans has not been previ- of what has been used in previous investigations of protein
ously examined under well-controlled experimental condi- metabolism in healthy human volunteers, with doses of ap-
tions. Comparisons with animal studies are problematic proximately 0.5 (21, 22) or 0.8 mg prednisone/kg䡠d (1, 19, 20)
because much higher doses have typically been used in ro- given for 5–7 d. Dexamethasone, which has higher potency
dents. Nevertheless, previous studies in rats have shown that than prednisone, was given orally at 8 mg/d for 4 d in two
high-dose administration of glucocorticoids for 5–12 d re- other investigations (3, 4). It is clinically established that
sults in pronounced skeletal muscle atrophy (up to 50% long-term glucocorticoid excess is associated with muscle
reduction in some muscles) and is accompanied by a reduced wasting and weakness (5–7), and it was reported that pred-
rate of synthesis of total mixed muscle proteins and myosin nisolone use for an average of 9 yr at 8 mg/d to treat rheu-
heavy chain (15, 16). These effects in rodents have been matoid arthritis was associated with reduced rate of synthe-
shown to be more prominent in fast-twitch, glycolytic mus- sis of muscle proteins (23). We therefore propose that either
cles (i.e. plantaris, gastrocnemius) than in oxidative muscles a higher dose or longer duration of prednisone administra-
(i.e. soleus), although the mechanism for such tissue speci- tion than was used in the current study is required to alter
ficity is not yet known (15, 16). postabsorptive protein metabolism in young healthy people.
In previous investigations in humans, whole-body protein This is line with rodent studies in which high doses of glu-
breakdown in the postabsorptive state was increased by cocorticoids have been used to demonstrate large rapid ef-
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fects on protein synthesis and breakdown (8 –16). However, role in skeletal muscle development, i.e. proliferation and
our aim was to use a dose and duration scheme that would differentiation, but its effects on muscle metabolism during
be relevant to the common clinical use of glucocorticoids for adulthood have not been established (45, 46).
short-term treatment of conditions such as inflammation or The large (44%) decline in circulating IGFBP-1 level during
asthma. The present study results indicate that there are no the prednisone trial is most likely due to the increase in
apparent disturbances in muscle protein metabolism, at least insulin, which has been shown to be a potent inhibitor of
in young healthy people. The threshold for glucocorticoid IGFBP-1 production (47). Previous studies reported that glu-
effects may differ with age or health status. For example, it cagon can stimulate production of IGFBP-1 (48, 49). How-
was reported that dexamethasone has more deleterious ef- ever, in the prednisone trial of the present study, the increase
fects on older rats (18 months), compared with younger rats in glucagon (12%) was much smaller than the increase in
(6 – 8 months) (10). The effect of glucocorticoids on protein insulin (109%), so any stimulatory effect of glucagon would
metabolism may also be less evident in the postabsorptive have been negated by the larger inhibitory effect of insulin.
state, compared with after a meal. Beaufrere et al. (19) showed Thus, insulin action (specifically on glucose metabolism) ap-
that following a gastrically infused meal, prednisone pre- pears to be diminished by prednisone treatment, but the
vented the normal increase in whole-body net balance of compensatory increase in insulin seems to reduce IGFBP-1
protein, apparently due to higher postmeal oxidation of Leu. levels. Consistent with our findings, Miell et al. (50) reported
The elevation of glucose, insulin, C-peptide, and glucagon in that after 3 d of treatment with dexamethasone (4 mg/d),
the present study suggests that carbohydrate metabolism normal male volunteers had increased levels of insulin and
may be more likely to be impaired by glucocorticoids in the IGF-I and decreased levels of IGFBP-1 and IGFBP-2. In that
postabsorptive state than protein metabolism (1– 4). study, though, there was also an increase in IGFBP-3 whereas
Prednisone use resulted in elevated insulin and IGF-I lev- IGF-II was unchanged, both of which differ from our results.
els. The rise in these two hormones may provide an anabolic Collectively the data suggest that changes in the IGF system
stimulus to counteract the glucocorticoid effect on protein during glucocorticoid treatment are mostly responses to the
metabolism. A major action of insulin on protein metabolism changing metabolic condition rather than direct effects of
is suppression of muscle protein breakdown (24, 25). Thus, glucocorticoids (47).
elevated insulin levels during prednisone administration Finally, there were almost no changes in muscle function
may have prevented an increase in protein breakdown in the in response to the short-term use of prednisone because
current study. This possibility is supported by reports that muscle strength and nearly all of the measures of muscle
insulin action on protein is maintained in both short-term mitochondrial function were unchanged. The one exception
prednisone users and patients with Cushing’s syndrome (21, was that mitochondrial ATP production in the presence of
22, 43). Arguing against this possibility, however, is the fact palmitoyl-l-carnitine was increased 12% during the pred-
that short-term glucocorticoid use can increase the rate of nisone trial, which suggests that the capacity to oxidize fatty
whole-body protein catabolism, whereas circulating insulin acids for fuel production in muscle was increased. To our
is also elevated (3, 4, 19, 21). There is also evidence that knowledge such a finding has not been previously reported.
glucocorticoids may actually blunt the ability of insulin to Prednisone had no significant effect on the activity of HAD,
suppress protein breakdown (1, 3). Thus, the interplay be- a key enzyme in the -oxidation pathway. The other mito-
tween insulin and glucocorticoids is not yet sufficiently re- chondrial enzymes and ATP production measurements
solved to reliably determine whether the rise in circulating tested, which are part of or share the common pathways of
insulin could have prevented some or all of the predicted the Krebs cycle and respiratory chain, were also unchanged.
effects of glucocorticoids in the current study. Thus, at least one other unique step in fatty acid transport or
Likewise, IGF-I has been reported to prevent the effects of oxidation was affected by prednisone to cause the increase
prednisone on protein metabolism when coadministered in ATP production with palmitoyl-l-carnitine. Carnitine
(100 g/kg䡠d) with prednisone (0.8 mg/kg䡠d) for 5 d in palmitoyl transferase is a likely candidate because it has been
young healthy subjects (44). A lower dose of IGF-I (80 g/ shown to be a rate-limiting step in fat oxidation (51). It should
kg䡠d) used in another study, however, did not effectively be noted, however, that despite the apparent increase in
alter the prednisone effect on protein metabolism (22). This muscle energy production from fat in response to prednisone
suggests that there may be a minimal level of IGF-I required administration, there was no change in the whole-body sub-
to counter the glucocorticoid effects on protein. Subjects in strate use during the study as measured by indirect calo-
the current study demonstrated an average increase of 35% rimetry. It is possible that shifts in fuel metabolism were
in plasma IGF-I after prednisone administration, which is localized only to muscle or that the muscle adaptations pre-
greater than previously reported under similar treatment ceded other steps in fat mobilization and transport required
conditions (20 –22). Because the levels of IGFBP-1 and to actually alter substrate use. These possibilities require
IGFBP-2 were decreased by 44 and 13%, respectively, in the further study.
prednisone trial, the bioavailability of IGF-I could be further In conclusion, the current study demonstrates that short-
increased. Thus, the elevated IGF-I may play a counterregu- term use of a moderate dose of prednisone has no effect on
latory role to maintain protein turnover at normal levels. The whole-body or leg muscle protein metabolism. There is also
6% reduction in circulating IGF-II in the prednisone trial no effect on muscle strength or muscle mitochondrial func-
could potentially dampen this stimulus, although the change tion. Circulating glucose and insulin levels are elevated in
in IGF-II is small in comparison with the increase in IGF-I. response to prednisone, indicating that glucose metabolism
Furthermore, IGF-II has been shown to play an important is more affected than protein metabolism by glucocorticoids.
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Acknowledgments 19. Beaufrere B, Horber FF, Schwenck WF, Marsh HM, Matthews D, Gerich JE,
Haymond MW 1989 Glucocorticoids increase leucine oxidation and impair
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Address all correspondence and requests for reprints to: K. S. Nair, 23. Gibson JN, Poyser NL, Morrison WL, Scrimgeour CM, Rennie MJ 1991
M.D., Ph.D., Mayo Clinic School of Medicine, Endocrinology Research Muscle protein synthesis in patients with rheumatoid arthritis: effect of chronic
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24. Meek SE, Persson M, Ford GC, Nair KS 1998 Differential regulation of amino
This work was supported by National Institutes of Health Grants
acid exchange and protein dynamics across splanchnic and skeletal muscle
RO1-DK41973 (to K.S.N.), T32-DK07352 (to K.R.S.), and MO1-RR00585. beds by insulin in healthy human subjects. Diabetes 47:1824 –1835
Additional support was provided by the Mayo Foundation and the 25. Nair KS, Ford GC, Ekberg K, Fernqvist-Forbes E, Wahren J 1995 Protein
Murdock-Dole Professorship (to K.S.N.) and the Mayo-Thompson Fel- dynamics in whole body and in splanchnic and leg tissues in type I diabetic
lowship (to K.R.S.). J.N. was supported by the Swedish Society of Med- patients. J Clin Invest 95:2926 –2937
icine, The Medical Research Council (09101), the Henning and Johan 26. Rooyackers OE, Balagopal P, Nair KS 1997 Measurement of synthesis rates
Throne-Holsts Foundation, and the Wenner-Gren Center Foundation. of specific muscle proteins using needle biopsy samples. Muscle Nerve Suppl
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