Am J Physiol Endocrinol Metab 289: E382–E390, 2005.
First published April 12, 2005; doi:10.1152/ajpendo.00591.2004.
Indinavir alters regulators of protein anabolism
and catabolism in skeletal muscle
Ly Q. Hong-Brown, Anne M. Pruznak, Robert A. Frost, Thomas C. Vary, and Charles H. Lang
Departments of Cellular and Molecular Physiology and Surgery,
Pennsylvania State University College of Medicine, Hershey, Pennsylvania
Submitted 13 December 2004; accepted in final form 5 April 2005
eukaryotic initiation factor 4E; eukaryotic initiation factor 4E-binding
protein-1; eukaryotic initiation factor 4G; mammalian target of rapamycin; ribosomal protein S6 kinase; muscle RING finger 1
UNTIL A CURE for human immunodeficiency virus (HIV) infection is found, antiretroviral therapy remains the first-line option
for the treatment of this patient population. Highly active
antiretroviral therapy (HAART) includes treatment with two
kinds of reverse transcriptase inhibitors and an HIV protease
inhibitor (11). This therapy decreases viral load and stimulates
immune function, thereby prolonging survival of patients with
AIDS. However, an increasing number of studies report that
patients receiving HAART develop a variety of metabolic
abnormalities (19). In particular, a majority of patients treated
with the protease inhibitor indinavir develop insulin resistance
Address for reprint requests and other correspondence: C. H. Lang, Dept.
Cell. Molec. Physiology, H166, Penn State College of Medicine, 500 Univ.
Dr., Hershey, PA 17033 (e-mail: [email protected]).
E382
(23, 44 – 47, 51) characterized by diminished insulin-stimulated glucose uptake in adipose tissue and muscle (51). Protease inhibitors also adversely affect lipid metabolism, as
exemplified by the elevated plasma concentrations of triglycerides and cholesterol as well as peripheral lipodystrophy and
increased central adiposity (3, 5, 25). Recently, various HIV
protease inhibitors have been reported to impair protein synthesis in cultured myocytes (22). However, the cellular mechanisms underlying this decrease in protein synthesis have not
been assessed under in vivo conditions.
Altered rates of muscle protein synthesis are often caused by
proportional changes in translation efficiency (8, 31, 53).
Translation of mRNA into protein is divided into three stages:
initiation, elongation, and termination. In many catabolic conditions, decreased muscle protein synthesis impairs mRNA
translation initiation, the first rate-determining phase of protein
synthesis (8, 28, 31, 35–37). Translation initiation is regulated
by a large number of protein factors termed eukaryotic initiation factors (eIFs). A critical point of translational regulation
involves binding of the 5⬘ end of cellular mRNA to the 43S
preinitiation complex, which is mediated by the cap-binding
protein complex eIF4F (47). The purpose of the present study
was to determine whether the acute administration of the
antiretroviral drug indinavir would decrease in vivo skeletal
muscle protein synthesis under basal conditions and, if so, to
determine whether this response was associated with defects in
signaling pathways known to regulate translation initiation.
Furthermore, because indinavir impairs insulin-stimulated glucose uptake, the ability of insulin to increase translation initiation was also investigated. Finally, catabolic states are often
associated with elevations in stress hormones, inflammatory
cytokines, and other negative effectors of muscle protein balance. Therefore, we also assessed whether indinavir increased
the circulating concentration of corticosterone, tumor necrosis
factor (TNF)-␣, interleukin (IL)-6, and insulin-like growth
factor-binding protein (IGFBP)-1, as well as the tissue level of
several ubiquitin ligases that are associated with increased rates
of muscle proteolysis.
MATERIALS AND METHODS
Animals. Male Sprague-Dawley rats weighing 200 –225 g were
purchased from Charles River Breeding Laboratories (Cambridge,
MA). Rats were acclimated for 1 wk in a light-controlled room
(12:12-h light-dark cycle) under constant temperature. Water and
standard rat chow were provided ad libitum. All experiments were
approved by the Institutional Animal Care and Use Committee of The
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. Section 1734 solely to indicate this fact.
0193-1849/05 $8.00 Copyright © 2005 the American Physiological Society
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Hong-Brown, Ly Q., Anne M. Pruznak, Robert A. Frost,
Thomas C. Vary, and Charles H. Lang. Indinavir alters regulators
of protein anabolism and catabolism in skeletal muscle. Am J Physiol
Endocrinol Metab 289: E382–E390, 2005. First published April 12,
2005; doi:10.1152/ajpendo.00591.2004.—The HIV protease inhibitor
indinavir adversely impairs carbohydrate and lipid metabolism,
whereas its influence on protein metabolism under in vivo conditions
remains unknown. The present study tested the hypothesis that indinavir also decreases basal protein synthesis and impairs the anabolic
response to insulin in skeletal muscle. Indinavir was infused intravenously for 4 h into conscious rats, at which time the homeostasis
model assessment of insulin resistance was increased. Indinavir decreased muscle protein synthesis by 30%, and this reduction was due
to impaired translational efficiency. To identify potential mechanisms
responsible for regulating mRNA translation, several eukaryotic initiation factors (eIFs) were examined. Under basal fasted conditions,
there was a redistribution of eIF4E from the active eIF4E 䡠 eIF4G
complex to the inactive eIF4E 䡠 4E-BP1 complex, and this change was
associated with a marked decrease in the phosphorylation of 4E-BP1
in muscle. Likewise, indinavir decreased constitutive phosphorylation
of eIF4G and mTOR in muscle, but not S6K1 or the ribosomal protein
S6. In contrast, the ability of a maximally stimulating dose of insulin
to increase the phosphorylation of PKB, 4E-BP1, S6K1, or mTOR
was not altered 20 min after intravenous injection. Indinavir increased
mRNA expression of the ubiquitin ligase MuRF1, but the plasma
concentration of 3-methylhistidine remained unaltered. These indinavir-induced changes were associated with a marked reduction in the
plasma testosterone concentration but were independent of changes in
plasma levels of IGF-I, corticosterone, TNF-␣, or IL-6. In conclusion,
indinavir acutely impairs basal protein synthesis and translation initiation in skeletal muscle but, in contrast to muscle glucose uptake,
does not impair insulin-stimulated signaling of protein synthetic
pathways.
INDINAVIR IMPAIRS PROTEIN SYNTHESIS
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transferred to PVDF membranes (Pall, Pensacola, FL). The blots were
blocked with 5% nonfat dry milk and incubated with polyclonal and
monoclonal antibodies. All antibodies were purchased from Cell
Signaling Technology (Beverly, MA) unless otherwise indicated.
These include antibodies that recognize the phosphorylated (p-) form
of PKB (Thr308), p-tuberin (Thr1462), p-S6 kinase (S6K1; Thr389,
Thr421/Ser424), p-eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1; Thr37/46), p-mamalian target of rapamycin (mTOR;
Ser2448), and p-S6 ribosomal protein (Ser235/236). Antibodies to total
PKB, mTOR, and the ribosomal protein (rp)S6 were obtained from
the same source, whereas the tuberin and S6K1 antibodies were from
Santa Cruz Biotechnology (Santa Cruz, CA). Unbound primary antibodies were removed by washing with TBS containing 0.05% Tween
20 and incubated with anti-rabbit or anti-mouse immunoglobulin
conjugated with horseradish peroxidase. Blots were incubated with an
enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK) and exposed to Kodak X-ray film (Rochester, NY).
The film was scanned (Microtek; ScanMaker IV, Los Angeles, CA)
and analyzed using NIH Image 1.6 software (NIH, Bethesda, MD).
For quantification of the eIF4E 䡠 eIF4G or eIF4E 䡠 4E-BP1 complexes,
supernatants were immunoprecipitated overnight with an anti-eIF4E
monoclonal antibody (kindly provided by Drs. L. S. Jefferson and
S. R. Kimball, Penn State College of Medicine, Hershey, PA).
Antibody-antigen complexes were collected using BioMag magnetic
beads (Qiagen, Valencia, CA). Beads were then washed in Trisbuffered saline (TBS) and proteins eluted using 2⫻ Laemmli sample
buffer. The precipitated material was examined by Western blot
analysis using total 4E-BP1, eIF4E, and eIF4G (Bethyl Laboratory,
Montgomery, TX), as described above (35–39).
Northern blot analysis of ubiquitin ligases. Total RNA was isolated
(Tri Reagent; Research Center, Cincinnati, OH) and 20 ␮g of total
RNA were run under denaturing conditions in 1.1% agarose-6%
formaldehyde gels using 1⫻ HEPES running buffer. Northern blotting
occurred via capillary transfer to Nytran SuPerCharge membranes
(Schleicher & Schuell, Keene, NH). Oligonucleotides for rat muscle
atrophy F-box (MAFBx; atrogin-1, 5⬘-CCCACCAGCACGGACTTGCCGACTCTCTGGACCAGCGTGC-3⬘) and muscle RING finger 1
(MuRF1; 5⬘-AGCGGAAACGACCTCCAGACATGGACACCGAGCCACCGCG-3⬘) were synthesized (IDT, Coralville, IA) and radioactively labeled using TdT (Promega, Madison, WI). For normalization of RNA loading, an 18S oligonucleotide was labeled by the same
method. Northern blots were hybridized using ULTRAhyb (Ambion,
Austin, TX). All membranes were initially washed twice in 2⫻
SSC-0.1% SDS for 5 min at 42°C and once in 0.2⫻ SSC-0.1%SDS
for 15 min at 42°C. Finally, membranes were exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA) and the resultant data quantified using ImageQuant software from Molecular Dynamics.
Plasma determinations. The plasma concentration of total testosterone (DPC, Los Angeles, CA), insulin (Linco Research, St. Charles,
MO), total insulin-like growth factor I (IGF-I) (37, 39), and corticosterone (DSL, Webster, TX) were determined by radioimmunoassay.
The 3-methylhistidine (3-MH) concentration in plasma was determined using reverse-phase HPLC after precolumn derivatization of
amino acids with phenyl isothiocyanate (37). The plasma concentrations of glucose and lactate were determined using a rapid analyzer
(Analox Instruments, Lunenburg, MA). The homeostasis model assessment (HOMA), which is defined as the fasting insulin concentration (␮U/ml) ⫻ fasting glucose concentration (mmol/l)/22.5, was used
as an index of insulin resistance (33). Plasma free fatty acid (FFA) and
triglyceride concentrations were also measured using colorimetric kits
(Wako Industrials, Osaka, Japan, and Sigma, St. Louis, MO, respectively). The IGFBP-1 content in plasma was determined by Western
blot analysis using commercially available antibodies (Upstate Biotechnology, Waltham, MA) (39). The plasma concentrations of
TNF-␣ and IL-6 were measured using a solid-phase sandwich enzyme-linked immunosorbent assay (BioSource International, Cama-
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Pennsylvania State University College of Medicine and adhered to
National Institutes of Health (NIH) guidelines for use of experimental
animals.
Experimental protocols. Rats were anesthetized with an intraperitoneal injection of ketamine and xylazine (90 and 9 mg/kg, respectively), and a jugular vein catheter was surgically implanted (37, 39).
Thereafter, rats were provided water ad libitum and fasted overnight.
The following morning, rats were administered a primed constant
intravenous infusion of indinavir (20 mg/kg ⫹ 0.25 mg 䡠 kg⫺1 䡠 min⫺1;
Merck, Rahway, NJ) or an equal volume of saline (1.5 ml/h) for a
period of 4 h. A primed constant infusion of indinavir was used to
maintain a stable plasma concentration of the drug, which is cleared
rapidly in rats (23). A comparable dosing regimen impairs insulinstimulated glucose uptake in muscle (23). At the conclusion of the 4-h
protocol, this infusion protocol produced a plasma indinavir concentration modestly higher than peak levels seen in human subjects taking
the drug orally (15).
Muscle protein synthesis and RNA and high-energy phosphate
content. In the first study, the in vivo rate of muscle protein synthesis
was determined using the flooding-dose technique (26, 37–39). Ten
minutes before the conclusion of the indinavir or saline infusion, rats
were injected intravenously with L-[2,3,4,5,6-3H]phenylalanine (Phe;
150 mM, 30 ␮Ci/ml; 1 ml/100 g body wt). Rats were then anesthetized with pentobarbital sodium, and 10 min after injection of Phe an
arterial blood sample was collected in a heparinized syringe from the
abdominal aorta for measurement of plasma cytokines and hormones
as well as plasma Phe specific radioactivity. Gastrocnemius muscle
was freeze-clamped in vivo, and tissues were powdered under liquid
nitrogen. Blood was centrifuged and plasma collected. All tissue and
plasma samples were stored at ⫺70°C until analyzed. A portion of the
powdered muscle was used to estimate the rate of incorporation of
[3H]Phe into protein (26, 37–39). To determine whether a change in
the number of ribosomes or the efficiency of mRNA translation was
responsible for the indinavir-induced changes in muscle protein synthesis, the RNA content of muscle was quantified. In striated muscle,
⬃85% of the RNA is ribosomal RNA, and, therefore, alterations in
total RNA content reflect changes in the number of ribosomes. Total
RNA content was measured from muscle homogenates (26). Translational efficiency was subsequently calculated by dividing the rate of
protein synthesis by the RNA content.
An aliquot of powdered gastrocnemius from control and indinavirtreated rats was extracted in cold perchloric acid, neutralized, and
used for the determination of adenosine triphosphate (ATP) and
creatine phosphate (CP) by standard fluorometric methods.
Indinavir-induced alterations in basal and insulin-stimulated signal transduction. In the second study, rats were infused with indinavir
and then challenged with intravenous insulin to determine whether
indinavir altered signal transduction pathways central to the regulation
of protein synthesis. Animals were treated as described above, except
that radiolabeled Phe was not injected. At the conclusion of the
infusion, rats were anesthetized with pentobarbitol sodium and injected intravenously with either insulin (5 U/kg) or an equal volume
(0.5 ml/rat) of saline. The gastrocnemius was collected 20 min later
and homogenized for measurement of protein factors important in the
control of mRNA translation. This insulin dose maximally stimulates
insulin receptor phosphorylation in skeletal muscle with nominal
activation of the IGF-I receptor (31, 35). The 20-min time point was
selected because it is the peak of the phosphorylation response for
essentially all of the protein factors determined in this study (31, 35).
Immunoprecipitation and Western blotting. Fresh gastrocnemius
was homogenized in a 1:4 ratio of ice-cold buffer (20 mM HEPES, pH
7.4, 2 mM EGTA, 50 mM NaF, 100 mM KCl, 0.2 mM ␣-glycerophosphate, 1 mM DTT, 0.1 mM PMSF, 1 mM benzamidine, 0.5 mM
sodium vanadate) (31, 35–39). The homogenates were centrifuged
and the supernatants diluted 1:1 with Laemmli sample buffer (50 mM
Tris, pH 6.8, 100 mM DDT, 2% SDS, 10% glycerol, 0.1% bromophenol blue). The samples were boiled, run on SDS-PAGE gels, and
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INDINAVIR IMPAIRS PROTEIN SYNTHESIS
rillo, CA). The plasma concentration of indinavir was measured by
reverse-phase HPLC (15).
Statistical analysis. Experimental data for each condition is summarized as means ⫾ SE, and the sample size is indicated in the figure
and table legends. In studies with two groups, data were analyzed
using a two-tailed pooled Student’s t-test. Statistical evaluation of the
data from studies with four groups was performed using ANOVA
followed by Student-Neuman-Keuls test to determine treatment effect
(Instat, San Diego, CA). Differences between the groups were considered significant when P ⬍ 0.05.
RESULTS
Fig. 1. Effect of indinavir on muscle protein synthesis. Protein synthesis was
measured by incorporation of [3H]phenylalanine (Phe) into proteins of gastrocnemius. On the day of the experiment, rats were infused iv with indinavir
for 4 h. Ten minutes before the end of the study, rats were administered
[3H]Phe, and gastrocnemius was excised and freeze-clamped. Values are
means ⫾ SE for 7– 8 animals per group. *P ⬍ 0.05 vs. values from timematched control animals infused with equal volume of saline.
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Insulin, ng/ml
Glucose, mmol/l
HOMA
Total IGF-I, ng/ml
Testosterone, ng/ml
Lactate, ␮mol/l
Triglyceride, mmol/l
FFAs, meq/l
Plasma corticosterone, ng/ml
Plasma IGFBP-1, AU
3-MH, ␮mol/ml
Control
Indinavir
0.95⫾0.19
6.1⫾0.2
7.3⫾0.9
748⫾39
3.1⫾1.0
0.78⫾0.02
0.48⫾0.04
0.65⫾0.12
85⫾6
1740⫾349
4.5⫾0.2
1.44⫾0.25
7.0⫾0.3*
12.9⫾2.1*
722⫾44
0.3⫾0.1*
0.67⫾0.06
0.60⫾0.09
0.59⫾0.08
91⫾4
4726⫾546*
4.3⫾0.2
Values are means ⫾ SE; n ⫽ 10 –12 per group. FFA, free fatty acids;
IGFGP-1, insulin-like growth factor-binding protein-1; 3-MH, 3-methylhistidine; AU, arbitrary units. Homeostasis model assessment (HOMA) for insulin
resistance was calculated as reported in Ref. 33. *P ⬍ 0.05 compared with
mean from time-matched saline-infused control group.
presents data on selected plasma hormone concentrations under
basal fasting conditions. Indinavir tended to increase the
plasma insulin concentration, but because of the variability this
change did not achieve statistical significance. The plasma
glucose concentration was significantly increased (⫹15%). As
a result, indinavir increased the HOMA insulin resistance
index by 75%. Indinavir did not alter the plasma IGF-I concentration but did reduce the testosterone concentration by
90%. There was no difference in the plasma concentrations for
corticosterone, lactate, FFAs, or triglycerides between control
and indinavir-treated rats.
Alterations in eIF4E distribution. The mechanistic interactions between indinavir and insulin were investigated by analyzing known regulatory steps in the control of translation
initiation (18, 47). Neither indinavir nor insulin altered the total
amount of eIF4E in gastrocnemius (Fig. 2). In contrast, indinavir increased the amount of eIF4E bound to 4E-BP1 (94%;
Fig. 2). Because the hyperphosphorylated ␥-isoform of 4E-BP1
cannot bind to eIF4E, the eIF4E immunoprecipitate contains
the two nonphosphorylated ␣- and ␤-isoforms of 4E-BP1 that
are resolved as a doublet on Western blot analysis. In salineinfused control rats, insulin decreased the inactive eIF4E䡠4EBP1 complex by 65%. Insulin also decreased eIF4E association with 4E-BP1 in indinavir-treated rats, and the value did
not differ from that of the control group administered insulin.
Indinavir decreased the amount of eIF4E bound to eIF4G by
80% under basal (e.g., nonstimulated) conditions (Fig. 3). In
contrast, insulin increased the amount of active eIF4E䡠eIF4G
complex a comparable extent in both control and indinavirtreated rats. To define the mechanism through which indinavir
modulates eIF4E availability the phosphorylation state of 4EBP1 was examined. Mitogen activation and amino acids stimulate multisite phosphorylation of 4E-BP1, leading to the
dissociation of 4E-BP1 from eIF4E and permitting the binding
of free eIF4E to eIF4G. Under basal conditions, indinavir
decreased the phosphorylation of 4E-BP1 by 70% (Fig. 4).
Conversely, the extent of 4E-BP1 phosphorylation was more
than doubled in muscle from control rats stimulated with
insulin. The incremental increase in the phosphorylation of
4E-BP1 induced by insulin in muscle from indinavir-treated
rats was comparable to that seen in control animals (Fig. 4).
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Indinavir-induced changes in muscle protein synthesis. Figure 1 (top) illustrates that indinavir decreased protein synthesis
in the gastrocnemius by 30%. There was no indinavir-induced
decrease in the total RNA per muscle protein (Fig. 1, middle),
indicating that the observed changes in muscle protein synthesis resulted from a corresponding change in the efficiency of
translation (Fig. 1, bottom) and not a change in the number of
ribosomes.
Muscle high-energy phosphate content. There was no difference in the ATP concentration in gastrocnemius between
control and indinavir-infused rats (7.11 ⫾ 0.12 vs. 7.22 ⫾ 0.24
␮mol/g wet wt, respectively). Likewise, there was no difference in CP levels between the two groups (20.6 ⫾ 1.1 vs.
19.9 ⫾ 1.3 ␮mol/g wet weight).
Plasma hormone, substrate, and indinavir concentrations.
At the conclusion of the 4-h infusion protocol the circulating
indinavir concentration averaged 19.7 ⫾ 3.7 ␮mol/l. Table 1
Table 1. Effect of indinavir on plasma concentrations
of hormones and substrates
INDINAVIR IMPAIRS PROTEIN SYNTHESIS
The interaction between eIF4E and eIF4G can also be
regulated, in part, by the phosphorylation of eIF4G, which is
enhanced by mitogen stimulation and inhibited by rapamycin
(43). Indinavir decreased the content of S1108-phosphorylated
eIF4G by 90% compared with values from control muscle (Fig.
5). The extent of eIF4G phosphorylation in muscle increased
almost twofold in control rats administered insulin. The incre-
Fig. 3. Effect of indinavir on association of eIF4G with eIF4E in skeletal
muscle. Indinavir was infused iv for 4 h, and then a maximally stimulating
dose of iv insulin was injected 20 min before tissue sampling. Top: eIF4E was
immunoprecipitated and amount of eIF4G bound to eIF4E assessed by immunoblotting. Middle: representative Western blot of total eIF4E. Bottom: densitometric analysis of immunoblots of eIF4G associated with eIF4E, where the
value from control rats infused with saline was set at 1.0 AU. Values are
means ⫾ SE; n ⫽ 7– 8 per group. Means with different letters are statistically
different from each other (P ⬍ 0.05).
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Fig. 4. Effect of indinavir on phosphorylation of 4E-BP1. Indinavir was
infused iv for 4 h, and then a maximally stimulating dose of iv insulin was
injected 20 min before tissue sampling. Phosphorylation of 4E-BP1 is ordered
and hierarchical, with Thr37 and Thr46 being the initial residues phosphorylated. Top: representative immunoblot for Thr37/46-phosphorylated 4E-BP1.
Bottom: densitometric analysis of phosphorylated 4E-BP1. Value from control
rats infused with saline was set at 1.0 AU. Values are means ⫾ SE; n ⫽ 7– 8
per group. Means with different letters are statistically different from each
other (P ⬍ 0.05).
ment in eIF4G phosphorylation in response to insulin was
comparable in control and indinavir-treated rats [1.1 vs. 0.9
arbitrary units (AU), respectively]; however, because the basal
value for the indinavir-treated rats was decreased relative to
control values, the value for the indinavir ⫹ insulin group
remained lower than that of the control ⫹ insulin group. The
changes in eIF4G phosphorylation were not due to a change in
the content of total eIF4G in muscle (Fig. 5).
S6K1 and S6 phosphorylation. The Ser/Thr protein kinase
referred to as the 70-kDa ribosomal protein S6 kinase 1
(p70S6K1 or S6K1) is also phosphorylated in response to
mitogens (12). There was little constitutive S6K1 phosphorylation in muscle from rats in the control ⫹ saline group (Fig.
Fig. 5. Effect of indinavir on the phosphorylation of eIF4G. Indinavir was
infused iv for 4 h and then a maximally-stimulating dose of iv insulin was
injected 20 min prior to tissue sampling. Top panels: a representative immunoblot of Ser1108-phosphorylated (P) and total eIF4G; bar graph: densitometric analysis of eIF4G phosphorylation where the value from control rats
infused with saline (Sal) was set at 1.0 AU. Values are means ⫾ SE; n ⫽ 7– 8
per group. Means with different letters are statistically different from each
other (P ⬍ 0.05).
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Fig. 2. Effect of indinavir on binding of eukaryotic initiation factor (eIF)4Ebinding protein-1 (4E-BP1) to eIF4E in skeletal muscle. Indinavir was infused
iv for 4 h, and then a maximally stimulating dose of iv insulin was injected 20
min before tissue sampling. Top: eIF4E was immunoprecipitated (IP), and
amount of 4E-BP1 bound to eIF4E assessed by immunoblotting (IB). Middle:
representative Western blot of total eIF4E. Bottom: densitometric analysis of
immunoblots of 4E-BP1 associated with eIF4E, where the value from control
rats infused with saline (SAL) was set at 1.0 AU (arbitrary units). Values are
means ⫾ SE; n ⫽ 7– 8 per group. Means with different letters are statistically
different from each other (P ⬍ 0.05).
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INDINAVIR IMPAIRS PROTEIN SYNTHESIS
6A). Indinavir did not appreciably change electrophoretic mobility of the bands, suggesting that the relative phosphorylation
of S6K1 was unaltered under basal conditions. Insulin decreased band mobility in control rats, and a comparable stimulation of S6K1 phosphorylation was observed after indinavir
treatment. Hierarchical multisite phosphorylation of residues in
the linker region (e.g., Thr389) leads to full and complete
activation of the kinase (20). There was a relative low level of
constitutive Thr389 phosphorylation under basal fasting conditions in control muscle (Fig. 6B). There was a marked increase
in S6K1 phosphorylation at the Thr389 site in muscle from
control animals administered insulin. A comparable increase in
Thr389 phosphorylation was observed in muscle from indinavir-treated rats injected with insulin. Insulin also stimulated the
phosphorylation of S6K1 at residue Thr421/Ser424 (e.g., residues in the autoinhibitory domain) to the same extent in both
control and indinavir-treated rats (data not shown).
The phosphorylation state of rpS6, a component of the 40S
ribosome and a physiologically relevant S6K1 substrate, exhibited a constitutive level of phosphorylation in muscle from
control rats (Fig. 6C). Basal phosphorylation of rpS6 was not
altered in muscle from indinavir-treated rats compared with
time-matched control rats. In response to insulin, S6 phosphorylation increased sixfold in both control and indinavir-treated
rats (Fig. 6C).
mTOR, tuberous sclerosis complex 2, and PKB phosphorylation. The proline-directed Ser/Thr protein kinase mTOR is a
common intermediate in the phosphorylation of both 4E-BP1
AJP-Endocrinol Metab • VOL
Fig. 7. Effect of indinavir on phosphorylation of mTOR (mammalian target of
rapamycin). Indinavir was infused iv for 4 h, and then a maximally stimulating
dose of iv insulin was injected 20 min before tissue sampling. Top and middle:
representative immunoblots of Ser2448-phosphorylated (P) and total mTOR,
respectively. Bottom: densitometric analysis of mTOR phosphorylation, where
the value from the control rats infused with saline was set at 1.0 AU. Values
are means ⫾ SE; n ⫽ 7– 8 per group. Means with different letters are
statistically different from each other (P ⬍ 0.05).
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Fig. 6. Effect of indinavir on phosphorylation of S6 kinase-1 (S6K1) and
ribosomal protein (rp)S6. Indinavir was infused iv for 4 h, and then a
maximally stimulating dose of iv insulin was injected 20 min before tissue
sampling. A: representative immunoblot for effect of indinavir on total S6K1.
B: representative immunoblot for phosphorylation of Thr389 site of S6K1. C:
representative immunoblot for phosphorylation of Ser235/236 of rpS6. D:
representative immunoblot of total rpS6. and Bottom: densitometric analysis of
S6 phosphorylation at Ser235/236, where the value from control rats infused
with saline was set at 1.0 AU. Values are means ⫾ SE; n ⫽ 7– 8 per group.
Means with different letters are statistically different from each other (P ⬍
0.05).
and S6K1 produced by amino acids and growth factors (18).
Indinavir decreased the constitutive phosphorylation of mTOR
(Ser2448) in muscle by 45% (Fig. 7). Insulin increased mTOR
Ser2448 phosphorylation to the same extent in muscle from
control and indinavir-treated rats. None of the treatments
altered the total amount of mTOR (Fig. 7).
One potentially important mechanism by which mTOR
might mediate the phosphorylation of S6K1 and 4E-BP1 in
response to nutrients and growth factors is by altering the
activity of the tuberous sclerosis complex (TSC), consisting of
the TSC1 (hamartin) and TSC2 (tuberin) proteins. Figure 8, A
and B, indicates that indinavir did not alter either the total
amount or the extent of Thr1462 phosphorylation of TSC2 in
skeletal muscle at the time point assessed. Insulin did not
acutely alter either total or phosphorylated TSC2 under in vivo
conditions. We cannot exclude the possibility that a transient
phosphorylation event was missed because only a single time
point was assessed for TSC2 phosphorylation. However, this
possibility appears unlikely, because phosphorylation of PKB
(Thr308), the upstream kinase responsible for TSC2 phosphorylation, was increased at the time point assessed (Fig. 8C).
Indinavir did not alter the ability of insulin to increase PKB
phosphorylation but did decrease constitutive PKB phosphorylation by 40% under basal conditions (control ⫽ 1.00 ⫾ 0.08
AU, indinavir ⫽ 0.59 ⫾ 0.09 AU, P ⬍ 0.05).
Potential negative regulators of muscle mass. The plasma
concentration of the inflammatory cytokines TNF-␣ and IL-6
were below the level of detection (⬍15 pg/ml) for both control
and indinavir-treated rats (data not shown). Table 1 shows that
indinavir increased the plasma concentration of IGFBP-1 almost threefold. A similar indinavir-induced increase was seen
for muscle MuRF1 mRNA content (control ⫽ 17 ⫾ 2 AU/18S
vs. indinavir ⫽ 69 ⫾ 7 AU/18S, P ⬍ 0.05), but there was no
detectable change in the expression of muscle MAFBx-1 (control ⫽ 96 ⫾ 8 vs. indinavir ⫽ 109 ⫾ 11 AU/18s, P ⫽ not
INDINAVIR IMPAIRS PROTEIN SYNTHESIS
significant). Finally, the plasma concentration of 3-MH, which
provides an indirect estimate of myofibrillar breakdown (13),
was not different between groups.
DISCUSSION
In the present study, we demonstrate that indinavir decreases
basal muscle protein synthesis by 30% but does not diminish
the ability of insulin to stimulate translation initiation. These
data are in contrast to those for glucose metabolism, where
indinavir does not alter basal glucose uptake but seriously
impairs insulin-stimulated glucose uptake in muscle (2, 6, 23,
40, 44 – 46, 51). Our results confirm the presence of an indinavir-induced insulin resistance related to glucose metabolism
as evidenced by the increased HOMA.
It is noteworthy that the indinavir-induced decrease in protein synthesis did not result from a reduction in energy stores
(as indexed by muscle ATP and CP content) or oxidative
metabolism (as indexed by lactate concentration). These findings are in contrast to the myopathy produced by other classes
of antiretroviral agents that results from inhibition of mitochondrial ATP-generating enzymes and produces hyperlactacidemia (9, 42). In addition, the reduced translational efficiency
was apparently not due to a generalized stress response, because the plasma corticosterone concentration was not altered.
Our data indicate that impaired translational efficiency, not
diminished capacity, is primarily responsible for the indinavirinduced decrease in protein synthesis. Other diseases and drugs
that inhibit translational efficiency function primarily by altering various protein factors that regulate peptide chain initiation
(28, 36 –39, 53). One of the rate-controlling steps in translation
initiation is the assembly of the eIF4F protein complex, composed of eIF4A, eIF4E, and eIF4G, that facilitates the binding
of mRNA to the 43S preinitiation complex. One of these protein
components, eIF4E, binds directly to the m7GTP cap structure
present at the 5⬘ end of the large majority of eukaryotic mRNA
AJP-Endocrinol Metab • VOL
and is critical to maintaining normal rates of protein synthesis.
During translation initiation, the eIF4E䡠mRNA complex binds
to eIF4G and eIF4A to form the active eIF4F complex. One
mechanism for modulating the formation of the eIF4F complex
is by altering the distribution of eIF4E between inactive and
active protein complexes. The assembly of the functional
eIF4F complex is controlled in part by 4E-BP1, which functions as a cap-dependent translational repressor (27, 32, 48).
This binding protein obstructs the interaction of eIF4G with
eIF4E and limits assembly of the active eIF4F complex (20).
Increased phosphorylation of 4E-BP1 in response to mitogens
results in the release of eIF4E, its binding to eIF4G, and
stimulation of mRNA translation (32, 48). Indinavir decreased
the phosphorylation of the translational repressor molecule
4E-BP1 under basal (e.g., no exogenous hormone stimulation)
conditions. This change was associated with an increased
amount of the inactive 4E-BP1䡠eIF4E complex and a reciprocal decrease in the amount of the active eIF4G䡠eIF4E complex.
The association of eIF4E with eIF4G may be rate limiting for
muscle protein synthesis (28, 35–39). Our results are consistent
with the observed decrement in basal muscle protein synthesis.
A decrease in the amount of eIF4E䡠eIF4G complex has also
been reported in cultured myocytes treated with indinavir, but,
in contrast to our in vivo findings, the response was independent of a change in 4E-BP1 phosphorylation (22).
Indinavir impaired the basal phosphorylation of protein
factors important in the regulation of peptide chain initiation,
including mTOR, 4E-BP1, and eIF4G. mTOR represents a
bifurcation point for the regulation of 4E-BP1 and S6K1
phosphorylation (18, 53). Therefore, it was unexpected that
indinavir decreased both mTOR and 4E-BP1 phosphorylation
but did not alter the constitutive phosphorylation of rpS6.
However, multiple factors modulate rpS6 phosphorylation, and
it is possible that an alterative pathway(s) is stimulated in
response to indinavir (55, 56). Regardless, these data suggest
that indinavir may not impair the translation of those mRNAs
containing a 5⬘-terminal oligopyrimidine tract. The indinavirinduced change in Ser1108 phosphorylation of eIF4G is noteworthy, because such a change has been associated with
enhanced rates of translation and may represent a secondary
mechanism regulating the interaction of eIF4E with eIF4G
(43). Although eIF4G phosphorylation is rapamycin sensitive
(e.g., mTOR dependent), the kinase action of mTOR on the
Ser1108 phosphorylation of eIF4G appears to be indirect (49).
The inhibition of constitutive phosphorylation of 4E-BP1,
eIF4G, and mTOR by indinavir was not due to a concomitant
reduction the plasma concentration of either insulin or IGF-I.
However, indinavir markedly decreased the circulating concentration of testosterone, a hormone well known for its
anabolic effects in skeletal muscle (14). Future studies will
need to determine whether this acute reduction in testosterone
contributes to the observed reduced phosphorylation of selected initiation factors and protein synthesis. A comparable
decrease in testosterone was not been reported in men taking
indinavir (7).
Elevations in insulin increase muscle protein synthesis under
in vivo conditions (1), in the perfused hindlimb (26), in
incubated muscles (58), and in cultured myocytes (22). However, because indinavir decreases insulin-stimulated glucose
uptake, we hypothesized a corresponding impairment of insulin-mediated mechanisms for controlling translation initiation.
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Fig. 8. Effect of indinavir on basal and phosphorylation of PKB and TSC
(tuberous sclerosis complex) 2 in skeletal muscle and in vivo conditions.
Indinavir was infused iv for 4 h, and then a maximally stimulating dose of iv
insulin was injected 20 min before tissue sampling. A and B: immunoblot of
Thr1462-phosphorylated (P) and total TSC2, respectively. C and D: immunoblot of Thr308-phosphorylated and total Akt/PKB, respectively. Blots are
representative of n ⫽ 7– 8 per group. Although there were no statistically
significant changes in either total or phosphorylated TSC2 in skeletal muscle
in response to any of the experimental perturbations, insulin increased Akt
phosphorylation in control rats at the same time point (control ⫹ saline ⫽
1.00 ⫾ 0.08 vs. control ⫹ insulin ⫽ 3.25 ⫾ 0.41 AU, P ⬍ 0.05). Insulininduced increase in Akt phosphorylation was not altered by indinavir administration (4.13 ⫾ 0.48 AU); however, indinavir alone did significantly decrease
basal Akt phosphorylation (control ⫹ saline ⫽ 1.00 ⫾ 0.08 vs. 0.59 ⫾ 0.09
AU, P ⬍ 0.05). Total Akt was not altered by any treatment.
E387
E388
INDINAVIR IMPAIRS PROTEIN SYNTHESIS
AJP-Endocrinol Metab • VOL
creases the mRNA content of MuRF1, but not atrogin-1, in
gastrocnemius muscle. Because of differences in the temporal
activation of these genes, it is possible that atrogin-1 would
also have been increased if the indinavir infusion had been of
longer duration. Despite the increase in MuRF1 mRNA, we did
not observe an elevation in the 3-MH concentration in blood.
Although it is recognized that measurement of protein breakdown based on 3-MH concentration and excretion has limitations (13), these data are consistent with the conclusion that
indinavir did not increase myofibrillar degradation at this early
time point. The effect of longer-term indinavir treatment on
protein balance, both synthesis and degradation, will need to be
assessed in future studies.
Our results demonstrate that indinavir depresses basal muscle protein synthesis, and this impairment appears mediated by
defects in mTOR and the formation of a functional eIF4F
complex but not a diminished activity of S6K1. In contrast to
the previously reported indinavir-induced insulin resistance
pertaining to glucose uptake (23, 44 – 47, 51), the ability of
insulin to stimulate translation initiation in muscle was not
altered by indinavir. The observed changes in the translational
control of protein synthesis were independent of defects in
energy production, as well as excess secretion of glucocorticoids and inflammatory cytokines, but were associated with a
reduction in testosterone. Hence, it is tempting to speculate that
therapeutic measures designed to enhance peripheral insulin
action in HIV-infected patients might also have the beneficial
consequence of improving muscle protein balance.
ACKNOWLEDGMENTS
We thank Drs. Leonard S. Jefferson and Scot R. Kimball (Pennsylvania
State College of Medicine) for the eIF4E antibody. Additionally, we thank
Danuta Huber and Nobuko Deshpande for expert technical assistance.
GRANTS
This work was supported in part by National Institutes of Health Grants
AA-11290 and GM-39277.
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