Departments of 1 Surgery and 2 Anesthesiology, University of Texas Medical Branch, Galveston, Texas 77550
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ABSTRACT |
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Testosterone administration (T) increases lean body mass and muscle protein synthesis. We investigated the effects of short-term T on leg muscle protein kinetics and transport of selected amino acids by use of a model based on arteriovenous sampling and muscle biopsy. Fractional synthesis (FSR) and breakdown (FBR) rates of skeletal muscle protein were also directly calculated. Seven healthy men were studied before and 5 days after intramuscular injection of 200 mg of testosterone enanthate. Protein synthesis increased twofold after injection (P < 0.05), whereas protein breakdown was unchanged. FSR and FBR calculations were in accordance, because FSR increased twofold (P < 0.05) without a concomitant change in FBR. Net balance between synthesis and breakdown became more positive with both methodologies (P < 0.05) and was not different from zero. T injection increased arteriovenous essential and nonessential nitrogen balance across the leg (P < 0.05) in the fasted state, without increasing amino acid transport. Thus T administration leads to an increased net protein synthesis and reutilization of intracellular amino acids in skeletal muscle.
fractional breakdown rate; amino nitrogen uptake
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INTRODUCTION |
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THE USE OF TESTOSTERONE to enhance athletic performance has long been practiced. However, the widespread use of anabolic steroids among the athletic community became an example of abuse (for review, see Ref. 15). For this reason, testosterone administration was often accompanied with negative connotations. More recently, however, clinical investigations have demonstrated the benefits of testosterone administration without the adverse side effects associated with uncontrolled pharmacological dosing. Restoration of testosterone levels in hypogonadal (3, 8, 17) and elderly (19) men has been shown to increase skeletal muscle protein synthesis and lean body mass (8). Administration in hypogonadal men (3, 8, 17) and to patients with muscular dystrophy (20) has also been demonstrated to increase lean body mass. A consistent finding of testosterone administration is the marked increase in muscle protein synthesis (8, 14, 19). However, muscle protein breakdown was not determined in these studies. Thus the effect of testosterone on net protein synthesis is yet unclear. A sustained increase in muscle protein synthesis could theoretically lead to an accrual of lean body mass if protein synthesis were continually greater than breakdown over time. Evidence for this is the demonstrated increase in lean body mass with testosterone administration alone (without the concomitant benefit of exercise) (2, 8).
Acute administration of other anabolic hormones has been demonstrated to increase skeletal muscle protein synthesis. Local infusion of insulin results in muscle anabolism by increased protein synthesis (6) or decreased protein breakdown (13). Local insulin-like growth hormone I (IGF-I) (13) and growth hormone (12) administration stimulates skeletal muscle protein synthesis, as well as a positive amino acid net balance. The stimulation of protein synthesis by these hormones appears closely related to an increased rate of inward amino acid transport (4). For example, intra-arterial insulin infusion into the leg increases inward amino acid transport (6) into skeletal muscle. Although not directly measured, the data suggest that IGF-I infusion also increases inward amino acid transport (9). We therefore hypothesized that increased protein synthesis with testosterone administration is accompanied by increased inward amino acid transport.
Because prolonged testosterone administration increases lean body mass (2, 8), we further hypothesized that testosterone increases net muscle protein synthesis. Hence, we investigated the effects of testosterone administration on muscle protein synthesis and amino acid balance 5 days after intramuscular injection. We evaluated these effects by using an established model (4, 5, 10) that enables the determination of tissue amino acid transport. In addition, we utilized an independent method (22) that enables the simultaneous evaluation of fractional synthetic and breakdown rates of skeletal muscle with biopsies of the vastus lateralis.
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METHODS |
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Subjects
Seven healthy males [28 ± 5 (SD) yr; 77 ± 8 kg; 177 ± 5 cm] were studied before and 5 days after testosterone enanthate injection. Written consent was obtained on all subjects, and the protocol was approved by the Institutional Review Board at the University of Texas Medical Branch at Galveston.Experimental Protocols
Subjects were admitted to the General Clinical Research Center (GCRC) and fasted from 2200 until completion of the isotope infusion study. At approximately 0700 the following morning, a 3-Fr 8-cm polyethylene catheter (Cook, Bloomington, IN) was inserted into the femoral vein and another into the femoral artery under local anesthesia. Both femoral catheters were used for blood sampling; however, the femoral arterial catheter was also used for indocyanine green infusion for the determination of leg blood flow. A 20-gauge polyethylene catheter (Insyte-W, Becton-Dickinson, Sandy, UT) was placed in an antecubital vein for infusion of labeled amino acids. A second 20-gauge polyethylene catheter was placed in the contralateral wrist and surrounded by a heating pad maintained at ~65°C for measurement of systemic concentration of indocyanine green.Baseline blood samples were obtained for the measurement of background
amino acid enrichment, indocyanine green concentration, and peak
testosterone concentration. Stable isotopes were concomitantly infused
at the following primed (PD) continuous infusion rates (IR) throughout
the 5-h study:
L-[ring-2H5]phenylalanine,
IR = 0.05 µmol · kg1 · min
1,
PD = 2 µmol/kg; L-[2-15N]lysine,
IR = 0.08 µmol · kg
1 · min
1,
PD = 7.2 µmol/kg;
L-[1-13C]leucine, IR = 0.08 µmol · kg
1 · min
1,
PD = 4.8 µmol/kg;
L-[1-13C]alanine, IR = 0.35 µmol · kg
1 · min
1,
PD = 35 µmol/kg. After 2 h of infusion (Fig.
1), a primed (2 µmol/kg) continuous (0.05 µmol · kg
1 · min
1)
infusion of L-[15N]phenylalanine
was initiated and maintained until the 4th h. The arterial and
intracellular
L-[15N]phenylalanine
enrichments at plateau and during the decay were utilized to determine
fractional breakdown rate (FBR).
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Biopsies of the vastus lateralis were performed as previously described (5) at 2 h, 4 h 30 min, 4 h 45 min, and 5 h of tracer infusion. Fractional synthetic rate (FSR) of skeletal muscle protein was determined by the incorporation of L-[ring-2H5]phenylalanine into protein from 2 to 4 h 45 min and also from 2 to 5 h (values averaged). The biopsies at 4 h 45 min and 5 h were utilized to determine FBR and averaged for data presentation.
Arteriovenous blood samples were drawn at 20-min intervals throughout
the last hour to determine amino acid kinetics. In addition, leg blood
flow was determined by indocyanine green infusion during this hour. To
measure leg blood flow, a continuous infusion (IR = 0.5 mg/min) of
indocyanine green was started 15 min before the kinetic hour.
Subsequent sampling was performed simultaneously from the femoral vein
and the heated wrist over this hour. Arterial samples for amino acid
kinetics were always taken after those from the femoral vein and wrist
to avoid interference with blood flow measurement. After each sampling,
indocyanine green infusion was uninterrupted for 10-15 min
before the next blood flow measurement. Arteriovenous samples were
taken at 4 and 5 h of the infusion protocol for the determination of
amino acid nitrogen balance. Blood was also sampled from the femoral
vein at 4 and 5 h for the determination of total testosterone over the
sampling period.
Immediately after the infusion protocol, the subject was fed and given an intramuscular injection of 200 mg of testosterone enanthate (Schein Pharmaceutical, Florham Park, NJ). On day 3, subjects returned to the GCRC at ~0700 for venous blood sampling to determine total testosterone concentrations. On day 5, the above protocol was repeated in the fasted state.
Analysis of Samples
Blood.
The concentrations of unlabeled phenylalanine, leucine, alanine, and
lysine, as well as the enrichment of their isotopic counterparts, were
simultaneously determined by gas chromatography-mass spectrometry (GC-MS) in blood by use of the internal standard approach (5). Whole
blood samples from the femoral vein and artery were immediately precipitated in preweighed tubes containing 15% sulfosalicylic acid
(SSA). A known internal standard mixture (100 µ/ml blood) was added
to the tube and thoroughly mixed. The composition of the internal
standard was as follows: 50.2 µmol/l of
L-[ring-13C6]phenylalanine,
249.0 µmol/l of
L-[2H4]alanine,
120.1 µmol/l of
L-[2H3]leucine,
and 182.0 µmol/l of
L-[2H4]lysine.
The tubes were reweighed for determination of blood volume and
centrifuged, and the supernatant was removed and frozen at
20°C until analysis. On thawing, 500 µl of the SSA extract was passed over a cation exchange column (Dowex AG 50W-8X, 100-200 mesh H+ form; Bio-Rad
Laboratories, Richmond, CA) and dried under vacuum with a speed vac
(Savant Instruments, Farmingdale, NY). To determine the enrichments of
the infused tracers and internal standards, the
nitrogen-acetyl-n-propyl esters were
prepared as previously described (4, 5). The isotopic enrichment of
free amino acids in blood was determined by GC-MS (model 5989, Hewlett-Packard, Palo Alto, CA) by chemical ionization and selected ion
monitoring (21). Data were expressed as a tracer-to-tracee ratio
(tracer/tracee), with correction for overestimation of enrichment
(skew) due to isotopomer distribution and for "overlapping"
contribution of isotopomers of small weight to the apparent enrichment
of isotopomers of greater mass (21).
Muscle.
Tissue biopsies of the vastus lateralis were immediately blotted and
frozen in liquid nitrogen. Samples were then stored at 75°C
until processed. On thawing, the tissue was weighed and protein was
precipitated with 0.5 ml of 10% perchloric acid. The tissue was then homogenized and centrifuged, and the supernatant was
collected. This procedure was repeated two more times, and the pooled
supernatant (~1.3 ml) was processed as the blood samples described in
Blood. To determine intracellular
enrichment of infused tracers, the
t-butyldimethylsylil derivative was
prepared as previously described (10) and analyzed by GC-MS (model
5989B, Hewlett-Packard) with electron impact ionization. Intracellular
enrichment was determined by correction for extracellular fluid based
on the chloride method (1).
Calculations
Kinetic model. Leg amino acid kinetics were calculated according to a three-pool compartment model that has been derived (4) and presented (5, 6, 10) previously. However, for simplicity and presentation of data, certain parameters are briefly described here.
Amino acids enter and leave the leg via the femoral artery (Fin) and femoral vein (Fout), respectively. Intercompartmental flow of free amino acids can occur between the artery (A), vein (V), and muscle (M). For example, FM,A refers to the net amino acid movement from the artery to the muscle, and FV,M refers to the movement from the muscle to the vein. These terms describe inward and outward tissue transport, respectively. Thus
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(1) |
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(2) |
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(3) |
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(4) |
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(5) |
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(6) |
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(7) |
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(8) |
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(9) |
FSR. Skeletal muscle FSR was calculated from the determination of the rate of tracer incorporation into the protein and the enrichment of the intracellular pool as the precursor
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(10) |
FBR. The derivation and assumptions of the calculation for FBR are described in detail elsewhere (22). Briefly, however, this new method for measuring fractional protein breakdown employs a variation of the traditional precursor-product method. In this case, the product is free intracellular amino acids, and the precursors are arterial blood and tissue protein. This model assumes that there is no label recycled from protein breakdown back to the free intracellular pool. Thus the arterial blood is the only source of tracer in the free intracellular pool, and both arterial blood and protein breakdown provide amino acids to the free intracellular pool. This model distinguishes the rate that amino acids are released from protein from the rate that amino acids are transported from plasma to tissue by use of the additional measurement of the steady-state enrichments of plasma and the free intracellular amino acids. With the systematic infusion of a labeled amino acid tracer to achieve isotopic equilibrium, the enrichment in the free intracellular pool is always lower than that in arterial blood. This is due to protein breakdown, which releases amino acids that dilute the isotope enrichment in the free intracellular pool. Therefore, the enrichment difference between arterial blood and the free intracellular pool reflects the fractional contributions of amino acids from these two pools. For instance, if the arterial enrichment is 0.10 and the intracellular free enrichment is 0.05, the fractional contributions from these two sources are 50% each.
After reaching an isotopic equilibrium of L-[15N]phenylalanine, the tracer infusion is stopped, and the decay curve of the free intracellular enrichment is decided by the arterial decay (which provides the tracer and a fraction of unlabeled amino acids) and FBR (which provides the rest of the unlabeled amino acids). Given a physiological steady state, FBR is constant, and the decay curves in the arterial and intracellular pools are measurable; therefore, FBR is measurable. The following equation was used for the calculation of FBR
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(11) |
Data Presentation and Statistical Analysis
Data are presented as means ± SE. Pre- and postinjection studies were compared by paired t-test with Bonferroni correction. (Family error rate) P ![]() |
RESULTS |
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Arterial steady-state tracer enrichment was achieved for all amino acid
tracers before and 5 days after injection (Table
1). Arterial enrichments were higher 5 days
after injection (Table 1; P < 0.05).
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Two days after testosterone (T) injection, total T concentrations increased to about twice the physiological range (2,094 ± 561 ng/dl; P < 0.05). Five days after the injection, however, total T returned to the upper physiological range (953 ± 283 ng/dl), although significantly greater than preinjection concentrations of 425 ± 99 ng/dl (P < 0.05).
T injection increased FSR from 1.60 ± 0.28 to 3.35 ± 0.38 %/day (mean increase 1.75 ± 0.40%/day;
P < 0.01), whereas the increase in
FBR was not quite significant (2.28 ± 0.11 to 2.64 ± 0.17 %/day; P = 0.06). Fractional net
balance (NB) increased from 0.68 ± 0.30 to 0.71 ± 0.39%/day (P < 0.05), with the
postinjection NB not different from zero. Because the calculation of
FBR is predicated on the assumption that no label is recycled from
protein breakdown to the free intracellular pool, it is possible that
the calculated FBR 5 days postinjection has a greater influence as a
result of previously "labeled" protein. However, the mean
intracellular free phenylalanine enrichment (tracer/tracee) was 0.0390 ± 0.0025, whereas the mean bound enrichment in the 5-h biopsy 5 days postinjection was 0.00067 ± 0.00019. Thus the contribution of
the protein-bound enriched phenylalanine could only account for less
than a 2% relative error in the FBR calculation.
Amino acid kinetics are presented in Table 2. Model calculations of protein synthesis (FO,M), as determined by phenylalanine disappearance from the intracellular free pool, demonstrated a significant increase after T, as did NB (Fig. 2). Because of the disproportionate representation of phenylalanine and lysine in skeletal muscle protein (5), values of FO,M and FM,O (in µmol/min) have been standardized for each amino acid. Model calculations of protein synthesis and breakdown were converted to milligrams per hour by assuming that phenylalanine and lysine represent 233 and 709 nmol/mg of dried muscle, respectively (5). Thus baseline FO,M yields similar values of 1,191 ± 356 mg/h for phenylalanine and 1,461 ± 208 mg/h for lysine (P = 0.52). Postinjection values increase to 2,634 ± 502 and 3,699 ± 1,105 mg/h for phenylalanine and lysine, respectively. Again, these values are not significantly different from each other (P = 0.40). Conversion of FM,O to protein breakdown equals baseline values of 1,842 ± 431 mg/h for phenylalanine and 1,801 ± 234 mg/h for lysine (P = 0.90) and postinjection values of 2,651 ± 516 mg/h for phenylalanine and 3,614 ± 1,008 mg/h for lysine (P = 0.42). Thus the apparent discrepancies in calculated protein synthesis (FO,M) and protein breakdown (FM,O) between the two essential amino acids that cannot be oxidized in skeletal muscle are reflective of their respective representations in skeletal muscle, which, when standardized, produce similar values for each parameter. Because leucine oxidation was not measured, calculated synthesis cannot be determined. However, conversion of baseline leucine FM,O [when assumption of 722 nmol/mg of dried muscle is made; (4)] again yields a similar value to phenylalanine and lysine (1,591 ± 251 mg/h; P = 0.35), which is consistent with other model calculations.
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After T, net balance (NB) values were significantly greater than
preinjection values for the essential amino acid tracers, although not
significantly greater than zero. Leucine NB also increased after
injection (22 ± 6 to
1 ± 6 nmol · min
1 · 100 ml leg
1;
P < 0.05), although it cannot be
determined whether this increase was related to an increased oxidation.
For a comparison of the arteriovenous model and direct methods, the
values of FO,M and FM,O
(nmol · min
1 · 100 ml leg volume
1) derived
from phenylalanine have been converted to FSR and FBR (%/h) on the
basis of the phenylalanine content in muscle [
233 nmol/mg
dried tissue (5)], the protein content in muscle [
25% (5)], and the assumption that muscle accounts for
60% of leg volume in normal subjects (9). Both methods resulted in essentially the
same value, with no statistical differences between direct calculation
(FSR/FBR) and converted values
(FO,M/FM,O
to FSR/FBR). Preinjection direct FSR and FBR were 0.067 ± 0.012 and
0.095 ± 0.005%/h, respectively, and the converted values for
FO,M and FM,O were 0.076 ± 0.022 and
0.11 ± 0.03%/h, respectively. Postinjection direct FSR and FBR
were 0.14 ± 0.016 and 0.11 ± 0.01%/h, respectively, and the
converted FO,M and
FM,O were 0.17 ± 0.03 and 0.17 ± 0.03%/h, respectively.
There was no evidence of increased tissue amino acid transport (Table 2) during the infusion protocol despite the finding that leg blood flow increased after T injection (333 ± 22 to 523 ± 92 ml/min; P < 0.05). Leucine transport was analogous to phenylalanine and lysine, because there was no change after injection. Because the calculation of leucine transport is not contingent on its fate in the cell (Eq. 3), it can be concluded that the inward transport of essential amino acids is not affected by T. There was, however, an increase in amino acid shunting (FV,A) from the artery to the vein after T injection.
Whole body appearance of phenylalanine decreased from 0.87 ± 0.10 µmol · kg1 · min
1
preinjection to 0.70 ± 0.07 µmol · kg
1 · min
1
postinjection (P < 0.05). Whole body
appearance of leucine also decreased (1.66 ± 0.08 to 1.47 ± 0.07 µmol · kg
1 · min
1;
P < 0.05), whereas lysine did not change.
T injection ameliorated the net efflux of amino acid nitrogen from the leg in the fasted state. Table 3 depicts the significant (P < 0.05) changes in essential and nonessential amino acid nitrogen balance across the leg.
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DISCUSSION |
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Testosterone exerts a strong anabolic effect in fasted skeletal muscle. Like acute infusions of insulin (6, 13), IGF-I (11, 13), and growth hormone (12), testosterone injection can ameliorate the negative amino acid balance of fasting. However, unlike these other hormones, testosterone administration does not affect inward amino acid transport to the tissue.
Acute insulin infusion increased inward amino acid transport, as measured by the three-compartment model utilized in the present study (6). In the present study, however, testosterone injection did not stimulate inward amino acid transport (FM,A; Table 2). The increased inward transport associated with local insulin infusion is accompanied by an increase in leg blood flow (6). However, this relationship is not characteristic of testosterone administration. The demonstrated 57% increase in blood flow was associated with an increased shunting of the essential amino acids phenylalanine and lysine (FV,A; Table 2). Thus, unlike insulin, where an increase in amino acid transport leads to an increase in protein synthesis, testosterone can increase protein synthesis without a concomitant increase in amino acid transport.
Because inward amino acid transport was not increased, intracellular amino acid availability for protein synthesis must be maintained by other mechanisms. During the fasted state, protein breakdown is normally much higher than protein synthesis (5, 6, 10) (Table 2, Fig. 2). Thus an ample supply of intracellular amino acids should be available to support a stimulation in protein synthesis. The strong relationship between protein breakdown and protein synthesis after testosterone injection (Fig. 3) supports the notion of increased efficiency of reutilization of amino acids from protein breakdown. Further support for increased reutilization is reflected by the changes in the ratios of outward transport to protein breakdown (FV,M/FM,O) and protein synthesis to protein breakdown (FO,M/FM,O). The ratio FV,M/FM,O decreased from 2.2 in the basal state to 1.3 (P < 0.05) 5 days after testosterone injection, whereas FO,M/FM,O increased from 0.67 to 1.0 (P < 0.02; Table 2). These changes indicate that testosterone injection results in a channeling of amino acids from protein breakdown back into protein, rather than releasing them to the blood. The change in amino acid nitrogen balance across the leg after injection (Table 3) provides further evidence of this reutilization. Nevertheless, extracellular amino acids must supply a certain portion of the precursors for synthesis after testosterone (FM,A is ~55% of RaM; Table 2). If this were not the case, it would not be possible for the muscle to be in zero balance, because there is a given rate of amino acid oxidation in the muscle. The absence of change in inward transport (FM,A) indicates that the basal rate was sufficient to supply the amino acids required to maintain zero balance. In addition, the absence of change in FM,A indicates that testosterone has limited effects on the splanchnic supply of amino acids to the periphery. The decrease in whole body appearance of phenylalanine and leucine further indicates that amino acids are not required from the splanchnic region, because whole body measures primarily reflect splanchnic metabolism in the fasted state (10).
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The increase in protein synthesis resulting from testosterone injection was capable of ameliorating the net amino acid efflux and protein catabolism of fasting, restoring each to a zero balance across skeletal muscle. For this metabolic state to continue, efficient reutilization of intracellular amino acids would also have to continue. Eventually an increase in protein turnover would be required to support continued synthesis in the muscle. Our data indicate that, after ~14 h of fasting, an increase in protein breakdown was forthcoming as direct FBR determination approached significance. However, fasting of this nature is normally limited, and subsequent feeding would provide an additional anabolic stimulus (amino acids and/or insulin) for continued protein synthesis (7). Over time, this continued net protein synthesis could lead to an accretion of lean body mass (2).
The findings of the present study are in accordance with previous studies indicating marked increases in muscle protein synthesis with testosterone administration (8, 14, 19). Our methodology allows a simultaneous determination of muscle protein breakdown that demonstrated an increase in net protein synthesis as the main result of testosterone administration. We utilized a testosterone dose that is typical of clinical replacement dosages (3, 14, 18, 19); however, the exact testosterone concentration required to evoke this response is uncertain. Although testosterone concentrations on the day of the infusion protocol (5 days postinjection) were in the upper physiological range, they were twice the physiological range 2 days (study day 3) after injection. Although a testosterone dose dependency is uncertain, a slightly supraphysiological dose used in the current study is associated with increased net protein synthesis.
We cannot rule out the possibility that an increase in testosterone has an indirect, or perhaps secondary, effect on the stimulation of protein synthesis. Testosterone enanthate administration has been shown to increase IGF-I concentrations in normal men (16). Although IGF-I levels were not measured in the present study, we have previously shown that the increase in protein synthesis with testosterone administration may be mediated by the stimulation of the intramuscular IGF-I system (19). Thus it is possible that an elevation of both testosterone and IGF-I is required, or that testosterone stimulates protein synthesis through a secondary system or hormone such as IGF-I.
In conclusion, testosterone injection resulted in an increase in net muscle protein synthesis that ameliorated the catabolism and net amino acid efflux of fasting. However, this increase in skeletal muscle protein synthesis was not supported by an increase in tissue transport of amino acids, but rather a reutilization of intracellular amino acids.
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ACKNOWLEDGEMENTS |
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The authors thank the nursing staff at the University of Texas Medical Branch General Clinical Research Center (GCRC) for invaluable assistance in the conduct of these studies, and Guy Jones and Zhanpin Wu for tireless efforts in sample analysis.
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FOOTNOTES |
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This study was conducted at the GCRC at the University of Texas Medical Branch at Galveston. It was funded by National Institutes of Health Grants M01-RR-00073 and DK-33952 and Shriners Hospital Grant 8940.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: A. A. Ferrando, Metabolism, 815 Market St., Galveston, TX 77550.
Received 25 February 1998; accepted in final form 3 August 1998.
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