Evaluation of signals activating ubiquitin-proteasome
proteolysis in a model of muscle wasting
William E.
Mitch,
James L.
Bailey,
Xiaonan
Wang,
Claudine
Jurkovitz,
David
Newby, and
S. Russ
Price
Renal Division, Emory University, Atlanta, Georgia 30322
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ABSTRACT |
The ubiquitin-proteasome proteolytic system is stimulated in
conditions causing muscle atrophy. Signals initiating this response in
these conditions are unknown, although glucocorticoids are required but
insufficient to stimulate muscle proteolysis in starvation, acidosis,
and sepsis. To identify signals that activate this system, we studied
acutely diabetic rats that had metabolic acidosis and increased
corticosterone production. Protein degradation was increased 52%
(P < 0.05), and mRNA levels encoding
ubiquitin-proteasome system components, including the
ubiquitin-conjugating enzyme E214k, were higher (transcription
of the ubiquitin and proteasome subunit C3 genes in muscle was
increased by nuclear run-off assay). In diabetic rats, prevention of
acidemia by oral NaHCO3 did not eliminate muscle proteolysis. Adrenalectomy blocked accelerated proteolysis and the rise in pathway mRNAs; both responses were restored
by administration of a physiological dose of glucocorticoids to
adrenalectomized, diabetic rats. Finally, treating diabetic rats with
insulin for
24 h reversed muscle proteolysis and returned pathway
mRNAs to control levels. Thus acidification is not necessary for these
responses, but glucocorticoids and a low insulin level in tandem
activate the ubiquitin-proteasome proteolytic system.
protein degradation; transcription; insulin; glucocorticoids
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INTRODUCTION |
THE PRINCIPAL EFFECT of insulin on protein metabolism
is to suppress protein degradation (9), and in insulin-dependent diabetic patients, insulin deprivation stimulates whole body protein degradation and amino acid oxidation (20, 21). Rats respond to insulin
deprivation in vivo by increasing protein degradation (27), and insulin
also suppresses protein degradation in cultured muscle cells (8, 10).
These results suggest that a low insulin level, or possibly insulin
resistance, could activate protein degradation in muscle with loss of
muscle protein and decrease in lean body mass. In fact, we found that
acute diabetes in rats produced by streptozotocin (STZ) injection
results in muscle atrophy due to accelerated protein degradation by the
ubiquitin-proteasome proteolytic pathway (22).
The ubiquitin-proteasome system is the major pathway accounting for the
turnover of muscle protein, and it is activated in several catabolic
conditions (18). Substrate proteins degraded by this system are first
marked by conjugation to ubiquitin in ATP-dependent reactions. The
ubiquitin-protein conjugates are then degraded by the 26S proteasome in
a process that unfolds the protein, releases ubiquitin, and degrades
the protein to small peptides and amino acids (6, 18). In acute
diabetes, as in other catabolic conditions, activation of this pathway
in muscle is associated with an increase in the content of mRNAs
encoding components of the pathway (18, 22). On the basis of results from a nuclear run-off experiment, we determined that at least the
higher level of ubiquitin mRNA in muscle of rats with chronic uremia or
acute diabetes is the result of an increase in gene transcription (3,
22).
Even though the ubiquitin-proteasome system is responsible for
degrading the bulk of protein in all cells, signals that activate this
proteolytic pathway are unclear. Certain stimuli have been associated
with activation of the ubiquitin-proteasome pathway in muscle. For
example, in normal rats or rats with chronic renal failure, we found
that acidification activates the ubiquitin-proteasome system (3, 17,
19). Another potential signal is an increase in glucocorticoids:
pharmacological doses increase muscle proteolysis (12), and
physiological levels are necessary but not sufficient for the catabolic
responses in rats with metabolic acidosis, starvation, or sepsis (16,
23, 28, 32). Acidosis, increased glucocorticoid production, or a low
level of insulin could function as signals activating muscle
proteolysis in acute diabetes. We investigated which of these stimuli
activate the ubiquitin-proteasome proteolytic system in muscle.
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METHODS |
Materials.
STZ was purchased from Pfanstiehl Laboratories (Waukegan, IL),
ZetaProbe GT membranes from Bio-Rad Laboratories (Hercules, CA),
[32P]dCTP and
[32P]CTP from Amersham
(Arlington Heights, IL), TriReagent from Molecular Research Center
(Cincinnati, OH), protamine-zinc-insulin (PZI) and
protamine-zinc-anulin from Anpro Pharmaceutical (Arcadia, CA), Humulin
R insulin from Eli Lilly (Indianapolis, IN), and Multistix 10 SG
reagent strips from Miles (Elkhart, IN). All other chemicals or
reagents were purchased from Sigma Chemical (St. Louis, MO). The
proteasome inhibitor MG-132 was generously provided by ProScript
(Cambridge, MA).
Rat model.
After anesthesia, 125- to 150-g, male Sprague-Dawley rats (Charles
River, MA) were given a tail vein injection of STZ (125 mg/kg prepared
fresh in 0.1 M citrate buffer, pH 4.0) and pair fed a 23% protein diet
with vehicle-injected, control rats, as described elsewhere (22). Rats
were housed in individual cages for the duration of the experiment and
studied 3 days (~72 h) after STZ injection. The only exception was
when we investigated how rapidly insulin would reverse muscle
proteolysis (see below). Urine was collected during the 24-h period
immediately before the experiments to measure corticosterone excretion
to assess glucocorticoid production. We used this method because
handling rats to obtain blood levels can acutely change the blood
corticosterone level, and the daily excretion rate yields an estimate
of the integrated, steady-state production rate (4, 13, 17).
To examine the influence of acidosis on muscle protein degradation,
control and STZ-treated rats were given a solution of NaHCO3 by gavage, as described
previously (24); other STZ-treated rats were given an equivalent amount
of sodium as NaCl throughout the experiment. Pair-fed control rats were
given NaHCO3 in a manner identical
to STZ-treated rats.
To determine the role of glucocorticoids in the proteolytic response in
muscle, 75-g rats underwent bilateral adrenalectomy (ADX) and were
given 0.077 M NaCl to drink. After a 10-day recovery period, ADX rats
were injected with STZ (125 mg/kg body wt) in the morning and were
given the 23% protein diet and 10% glucose-0.077 M NaCl to drink ad
libitum for 24 h. Subsequently, they were given 0.077 M NaCl to drink
ad libitum. Another group of ADX-STZ rats was given dexamethasone (2 µg · 100 g body
wt
1 · day
1
sc) in two equal injections starting on the day of STZ injection. Control ADX rats were treated in a similar fashion, except they did not
receive STZ. ADX-control and ADX-STZ rats were pair fed to ADX-STZ rats
receiving dexamethasone and studied 3 days (~72 h) after STZ injection.
To determine that the proteolytic response in muscles of STZ-treated
rats is due to insulin insufficiency, STZ-treated rats were given a
daily injection of the long-acting bovine PZI (8 U/100 g body wt sc)
beginning on the morning after receiving STZ (day
1) and on the subsequent morning
(day 2). Rats were fasted the night
before muscles were isolated for measurements of protein degradation
and levels of mRNAs encoding components of the ubiquitin-proteasome pathway (day 3).
To examine whether the proteolytic response in diabetic rats could be
reversed by insulin, rats were injected with STZ and pair fed with
control, sham-injected rats. The diabetic rats were not treated with
insulin for the initial 3 days, and on the morning of
day 3 they were randomly divided into
two groups: 1) STZ-treated rats that
received the shorter-acting Humulin R insulin (2.5 U/100 g body wt) and
the longer-acting bovine protamine-zinc-anulin insulin (0.5 U/100 g
body wt) to have a sustained action of insulin and
2) STZ-treated rats that did not
receive insulin. Pair feeding was continued during the day; after an
overnight fast, muscles were isolated and studied.
Measurement of muscle protein degradation.
The mixed-fiber epitrochlearis muscle was studied because it exhibits
rates of protein turnover in the presence or absence of insulin that
are similar to those measured in the bulk of muscle in adult rats (5).
Epitrochlearis muscles were dissected from diabetic and control rats
and preincubated for 30 min at 37°C in Krebs-Ringer bicarbonate
media containing 10 mM glucose and 0.5 mM cycloheximide and
equilibrated with 95% O2-5%
CO2 (pH 7.4) (3, 19, 22). The
muscles were then placed in fresh media, regassed, and incubated for 2 h at 37°C. At the end of the incubation period, TCA (final
concentration 10%) was added to precipitate proteins. Free tyrosine in
the media was measured to calculate the rate of protein degradation,
because we have found that tyrosine does not accumulate in the
intracellular space in muscles of diabetic or control rats treated with
inhibitors of proteolytic pathways (22). To evaluate changes in the
activity of specific proteolytic pathways, inhibitors were added as
described in earlier studies (3, 19, 22).
Northern blot hybridizations and nuclear run-off assay.
RNA was isolated from the gastrocnemius muscles using
TriReagent and separated in a formaldehyde-agarose gel by
electrophoresis before transfer to a ZetaProbe GT membrane.
Hybridizations were performed as described previously (22). The
gastrocnemius muscle was studied, because it is a mixed-fiber muscle
and changes in protein turnover reflect those occurring in the
epitrochlearis muscle (5), and levels of mRNAs encoding components of
the ubiquitin-proteasome pathway are coordinated with activation of this pathway in rats with acidosis, chronic renal failure, or starvation (3, 19, 22, 23).
To perform nuclear run-off assays, nuclei were isolated from
hindquarter muscles from each rat. Transcription measurements were as
described previously (3, 22).
Statistical analysis.
Values are mean ± SE. Results were analyzed by using the
Student's paired t-test when results
from two experimental groups were compared or by using ANOVA when data
from more than two groups were studied. For data analyzed by ANOVA,
pairwise comparisons were made by the Student-Newman-Keuls test.
P < 0.05 was considered significant.
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RESULTS |
Acidosis and muscle proteolysis.
Previously, we found that metabolic acidosis in otherwise
normal rats stimulates the ubiquitin-proteasome pathway in muscle (19).
Because STZ-treated rats develop acidosis (22), acidification could
be the stimulus accelerating muscle protein degradation. To examine
this possibility, rats were given oral
NaHCO3 for 3 days (~72 h) after
the STZ injection to prevent acidemia. The serum
HCO3 concentration was not
different between pair-fed, control, and STZ-treated rats given
NaHCO3 but was lower in
STZ-treated rats given NaCl. Giving acutely diabetic rats
NaHCO3 did not correct their
hyperglycemia (Table 1).
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Table 1.
Serum HCO3 and blood glucose values for control rats,
acutely diabetic rats, and acutely diabetic rats given
NaHCO3
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Protein degradation rates in muscles from
STZ-treated or STZ-HCO3 rats were
greater than the rate measured in control rat muscles (Fig.
1). When inhibitors of lysosomal and
calcium-dependent proteolysis were added, protein degradation rates
measured in muscles of STZ-treated rats or
STZ-HCO3 rats were greater than the rates measured in muscles of control rats (Fig. 1). These results
indicate that the ubiquitin-proteasome system must be responsible for
the accelerated protein degradation.

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Fig. 1.
Acidosis is not a stimulus of ubiquitin-proteasome-dependent protein
degradation in muscles of acutely diabetic rats.
Protein degradation was measured in isolated epitrochlearis muscles of
pair-fed, control rats given oral
NaHCO3 (CTL), acutely diabetic
rats given oral NaHCO3
[streptozotocin (STZ) + HCO3], or diabetic rats
given oral NaCl (STZ). Studies were begun 3 days after STZ injection.
One muscle from each rat was incubated without proteolytic inhibitors
(solid bars); the contralateral muscle was incubated in the presence of
lysosomal and calcium-dependent proteolytic inhibitors (overlapping
open bars). * P < 0.05 vs.
control muscle protein degradation under same incubation conditions
(n = 6 for each group).
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When acutely diabetic rats were given
NaHCO3, the level of ubiquitin
mRNA in mixed-fiber gastrocnemius muscle was as high as in muscle of
untreated, acutely diabetic rats and was 147% (P < 0.05) greater than the level in
muscles of control rats (Fig. 2).
Similarly, the levels of mRNAs encoding the C3 and C9
-type proteasome subunits as well as the C5
-type subunit were higher in
muscle of diabetic rats and diabetic rats given
NaHCO3 than in control rat muscles
(Fig. 2). The levels of the 1.2-kb mRNA of the
E214k ubiquitin-conjugating enzyme
in STZ-treated or STZ-HCO3 rat
muscles were also higher than in control rats. The level of the 1.8-kb
E214k was higher in diabetic rats
given NaHCO3 (Fig. 2). The reason
for the increased level of the 1.8-kb mRNA in muscle of STZ-treated
rats given NaHCO3 is unknown.
These data, together with the muscle proteolysis measurements, indicate
that activation of the ubiquitin-proteasome pathway by acute diabetes
is not dependent on acidemia.

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Fig. 2.
Acidosis is not a stimulus that increases muscle levels of ubiquitin
(Ub)-proteasome system mRNAs in acute diabetes. RNA was isolated from
gastrocnemius muscles of pair-fed control rats given oral
NaHCO3 (CTL), acutely diabetic
rats given oral NaHCO3
(STZ-HCO3), or diabetic rats
given oral NaCl (STZ). Studies were begun 3 days after STZ injection.
A: Northern blots were hybridized with
cDNAs for chicken ubiquitin, rat
E214k ubiquitin-conjugating
enzyme, and rat C3, C5, and C9 proteasome subunits; corresponding 18S
and 28S rRNA bands are also shown. B:
RNA band intensities were quantified, and results from 4 rats were
assessed. Values are normalized using 28S rRNA values and are expressed
as percentage (mean ± SE) of mean control rat value. Solid bars,
diabetic rats given NaHCO3; open
bars, diabetic rats given NaCl.
* P < 0.05 vs. control.
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Glucocorticoids and muscle proteolysis.
Corticosterone excretion was significantly
(P < 0.001) less in pair-fed,
control rats than in diabetic rats (Fig.
3), indicating that glucocorticoid
production is increased by acute diabetes as it is in patients (25,
26). To evaluate the role of glucocorticoids in the proteolytic
response to acute diabetes, we studied ADX rats. Results from
ADX-control rats were compared with rates measured in muscle of ADX
rats given STZ with (ADX-STZ-GC) or without (ADX-STZ) dexamethasone at
a dose that approximates physiological glucocorticoid levels (16, 17).
After an overnight fast, blood glucose levels in ADX-control, ADX-STZ,
and ADX-STZ-GC rats were 51 ± 11, 160 ± 45, and 236 ± 26 mg/dl, respectively. Protein degradation was higher in muscles of
ADX-STZ-GC rats replaced with glucocorticoids than in muscles of
ADX-control or ADX-STZ rats that were not treated with glucocorticoids
(Fig. 4). When lysosomal and
calcium-dependent proteolysis inhibitors were present in the incubation
media, proteolysis remained higher in muscles of ADX-STZ-GC rats than
in ADX-STZ or ADX-control rats (Fig. 4).

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Fig. 3.
Corticosterone production is increased by acute diabetes. Urine was
collected for 24 h, and total daily excretion of corticosterone was
measured. Experimental collections were performed between
days 2 and
3 in control (CTL) or diabetic rats
(STZ) 3 days after STZ injection. Excretion rates for individual rats
are shown. Mean (+) value in 8 STZ rats was higher
(P < 0.001) than in 8 control
rats.
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Fig. 4.
Glucocorticoids are required for activation of muscle protein
degradation by ubiquitin-proteasome system. Protein degradation was
measured in isolated epitrochlearis muscles of 3 groups of
adrenalectomized (ADX) rats: pair-fed control ADX rats (ADX), ADX
diabetic rats (ADX + STZ), or ADX diabetic rats given dexamethasone
(ADX + STZ + GC). Studies were begun 3 days after STZ injection. One
muscle from each rat was incubated without proteolytic inhibitors
(solid bars); the contralateral muscle was incubated in the presence of
lysosomal and calcium-dependent proteolytic inhibitors (overlapping
open bars). * P < 0.05 vs.
control muscle protein degradation under same incubation conditions
(n = 6).
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Ubiquitin mRNA in muscles of ADX-control and diabetic ADX rats did not
differ and were lower (P < 0.05)
than in muscles of diabetic ADX rats given glucocorticoids (Fig.
5). Similarly, levels of mRNAs encoding
other pathway components were significantly lower
(P < 0.05) in muscle of ADX-control
and ADX-STZ rats than ADX-STZ-GC rats (Fig. 5).

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Fig. 5.
Glucocorticoids are required for increased muscle levels of
ubiquitin-proteasome system mRNAs. RNA was isolated from gastrocnemius
muscles of 3 groups of ADX rats: ADX, ADX + STZ, and ADX + STZ + GC.
Studies were begun 3 days after STZ injection.
A: Northern blots were hybridized with
cDNAs for chicken ubiquitin, rat
E214k ubiquitin-conjugating
enzyme, and rat C3, C5, and C9 proteasome subunits.
B: RNA band intensities were
quantified, and results from 4 rats were assessed and are expressed as
in Fig. 2. Solid bars, ADX-STZ rats; open bars, ADX + STZ + GC rats.
* P < 0.05 vs. control.
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Insulin and muscle proteolysis.
We gave STZ rats daily doses of long-acting PZI beginning on the
morning after the STZ injections (days
1 and 2). After an overnight fast and ~24 h after the last insulin injection, blood glucose concentrations in control and diabetic rats on
day 3 were 116 ± 8 and 266 ± 49 mg/dl, respectively (P < 0.05 vs.
control); blood glucose in diabetic rats given insulin was 330 ± 10 mg/dl (P < 0.05 vs. control).
Corticosterone excretion during the 24 h after the last
insulin injection and before muscles were isolated to measure protein
degradation was 2.13 ± 0.20 µg/kg body wt in control
rats, 3.18 ± 1.09 µg/kg body wt in diabetic rats receiving insulin (not significant vs. control rats), and 9.79 ± 3.36 µg/kg body wt in diabetic rats (P < 0.05 vs. control or diabetic rats treated with insulin). Insulin prevented
the accelerated rate of proteolysis in muscle. We attribute this result
to suppression of proteolysis by the ubiquitin-proteasome system for
two reasons: 1) protein degradation
remained elevated in muscle of acutely diabetic rats, even though
lysosomal and calcium-activated proteases were blocked (Fig.
6), and
2) when the proteasome inhibitor
MG-132 was added, protein degradation in muscles from acutely diabetic rats was not different from the rate measured in muscle of control rats
or diabetic rats receiving insulin (Fig. 6).

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Fig. 6.
Insulin blocks proteolytic response in diabetic rats. Protein
degradation was measured in isolated epitrochlearis muscles of pair-fed
control rats (CTL), diabetic rats given insulin (STZ + insulin), or
diabetic rats (STZ). Insulin treatment was begun on day after STZ
injection, and protein degradation was measured on day
3 after STZ injection. One muscle from each rat was
incubated with inhibitors of lysosomal and calcium-dependent
proteolysis (solid bars); the contralateral muscle was incubated in the
presence of lysosomal and calcium-dependent proteolytic inhibitors plus
the proteasome inhibitor MG-132 (overlapping open bars).
* P < 0.05 vs. control muscle
protein degradation under same incubation conditions
(n = 6 for each group).
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mRNA levels were higher (P < 0.05)
in muscles of diabetic rats than in muscles of control, pair-fed rats,
but when diabetic rats were given insulin for 3 days the levels of
these mRNAs were not different from those measured in muscles of
control rats (Fig. 7).

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Fig. 7.
Insulin prevents increase in levels of ubiquitin-proteasome system
mRNAs in muscles of diabetic rats. RNA was isolated from gastrocnemius
muscles of CTL, STZ + insulin, and STZ.
A: Northern blots were
hybridized with cDNAs for chicken ubiquitin, rat
E214k ubiquitin-conjugating
enzyme, and rat C3, C5, and C9 proteasome subunits.
B: RNA band intensities were
quantified, and results (n = 4 rats)
are expressed as in Fig. 2. Solid bars, diabetic rats given insulin;
open bars, untreated diabetic rats.
* P < 0.05 vs. control.
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We also examined whether insulin would reverse the increase in muscle
protein degradation and reduce the higher levels of mRNAs in muscle of
diabetic rats. On day 4 after STZ
injection the diabetic rats were arbitrarily divided into two groups
that were pair fed: 1) diabetic rats
that received insulin and 2)
diabetic rats that did not receive insulin. Insulin treatment for 12 h did not give a consistent pattern of changes in muscle protein degradation. Insulin treatment for 24 h led to a blood glucose of 138 ± 29 vs. 132 ± 16 mg/dl in control rats and 247 ± 14 mg/dl in acutely diabetic rats. The basal rates of protein degradation in
muscles of control and acutely diabetic rats given insulin for 24 h
were not different statistically (152.8 ± 5.8 vs. 156.1 ± 11.5 ng
tyrosine · g
1 · h
1,
respectively). Both values were lower than the rate measured in muscle
of diabetic rats (261.1 ± 11.6 ng
tyrosine · g
1 · h
1,
P < 0.05). Ubiquitin and proteasome
C3 subunit mRNA values in muscles of diabetic rats given insulin were
also reduced to control levels (data not shown).
Proteasome subunit gene transcription.
To evaluate whether the higher level of proteasome subunit mRNAs is
related to increased gene transcription, nuclei were isolated from
muscles of control and STZ-treated rats, and run-off assays were
performed. A representative result (Fig. 8)
indicates that transcription of the genes encoding the C3 proteasome
subunit as well as ubiquitin is increased in muscles of STZ rats
compared with control rats. In three pairs of rats with acute diabetes and their pair-fed controls, diabetes was associated with increased transcription of the C3 proteasome subunit and ubiquitin genes of 78 ± 12 and 58 ± 10%, respectively
(P < 0.05 vs. pair-fed control rats). Transcription of the glyceraldehyde 3-phosphate dehydrogenase gene was unchanged by diabetes.

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Fig. 8.
Acute diabetes stimulates transcription of the proteasome C3 subunit
gene. Nuclei from muscles of pair-fed control (CTL) or diabetic (STZ)
rats were isolated, and nuclear run-off assays were performed to
measure relative transcription rates of glyceraldehyde 3-phosphate
dehydrogenase (GAPDH), proteasome C3 subunit, or ubiquitin genes.
Studies were begun 3 days after STZ injection. Representative results
(from 3 pairs) of muscle nuclei of 1 diabetic and 1 pair-fed control
rat are shown.
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DISCUSSION |
In adult humans the principal effect of insulin on protein metabolism
is suppression of protein degradation (9, 15). This also appears to be
true in rats, because we found that an acute decrease in insulin
production causes substantial loss of body weight, reduced muscle,
liver, and adipose tissue mass, and accelerated muscle proteolysis via
the ubiquitin-proteasome system (22). Potential signals initiating this
proteolytic response could be metabolic acidosis and/or an increase in
glucocorticoids, since metabolic acidosis induced by feeding
NH4Cl increases the steady-state
production of glucocorticoids and stimulates muscle protein
degradation, whereas high doses of glucocorticoids stimulate muscle
protein degradation, at least transiently (1, 12). Finally, the
proteolytic response could be due to a decrease in insulin level, which
accelerates protein degradation, because the normal, insulin-related
suppression of muscle proteolysis is diminished (5).
We excluded metabolic acidosis as a prominent stimulus of muscle
protein degradation caused by a low insulin level. When we gave
NaHCO3 to acutely diabetic rats,
the accelerated rate of muscle protein degradation was unchanged in
muscle, even though serum bicarbonate was normal. Likewise, preventing
acidosis did not attenuate the rise in levels of mRNAs encoding
components of the ubiquitin-proteasome pathway. Thus the acidosis of
acute diabetes does not activate the proteolytic system in muscle,
although acidification will lower the pH in cultured muscle cells and
stimulate protein degradation (8, 11). This was unexpected in light of
our finding that correction of acidosis in chronically uremic rats
blocks the increase in muscle proteolysis and the rise in ubiquitin and
proteasome component mRNAs (3). The mechanism for activation of this
system by acidemia is complicated, however, because we found that
induction of metabolic acidosis in normal rats by feeding
NH4Cl lowers the pH in rat muscle
(measured by NMR) by only a small amount, whereas the acidosis of
chronic renal failure does not change muscle pH or the recovery of
muscle pH after intracellular acidification resulting from muscle
contraction (2). It is difficult to implicate changes in muscle pH as a primary signal activating the ubiquitin-proteasome system in muscle.
In normal rats the proteolytic response to acidification in muscle
requires glucocorticoids (16, 17, 23). The present results demonstrate
that glucocorticoids are also required for activation of the
ubiquitin-proteasome pathway in acutely diabetic rats. First, urinary
corticosterone excretion, a measure of the integrated rate of
glucocorticoid production, is high in these rats (17). Second, ADX
prevented the accelerated protein degradation in muscles of acutely
diabetic rats, and the proteolytic response was restored when the ADX
diabetic rats were given dexamethasone at a dose that yields
physiological replacement levels. Moreover, the pattern of changes in
the levels of mRNAs of the ubiquitin-proteasome pathway in muscle was
consistent with the changes in muscle proteolysis (23). We did not
evaluate the proteolytic or mRNA responses in normal (nondiabetic) ADX
rats given the same amount of dexamethasone, because we have found that
this dose of dexamethasone or one slightly higher does not increase
muscle proteolysis or the levels of ubiquitin-proteasome pathway mRNAs
(16, 17, 23). Thus the results we obtained in control and ADX diabetic
rats are consistent with the conclusion that two stimuli, i.e.,
glucocorticoids and a low insulin level, are required to stimulate the
ubiquitin-proteasome pathway.
The critical role of insulin in controlling muscle proteolysis was
demonstrated by giving insulin to rats after STZ administration. Even
though blood glucose was not corrected, insulin administration prevented the increase in muscle proteolysis and the rise in
ubiquitin-proteasome pathway component mRNAs. These results suggest
that the beneficial influence of insulin is independent of the blood
glucose. Moreover, even when the ubiquitin-proteasome system is
activated by acute diabetes, accelerated muscle proteolysis was reduced
to a normal level after only 24 h of insulin. Likewise, insulin reduced
the levels of mRNAs encoding components of the ubiquitin-proteasome system in muscle to levels measured in control rats. On the other hand,
insulin did suppress glucocorticoid production. Again, these results
point to a dual role of low insulin plus glucocorticoids as the signal
activating the ubiquitin-proteasome pathway in muscle.
Interestingly, the rate of muscle proteolysis and levels of
ubiquitin-proteasome pathway mRNAs were regulated in a coordinated fashion. In studies of ADX rats with diabetes or diabetic rats given
insulin, a change in protein degradation was accompanied by a parallel
change in mRNA levels. Thus we could not determine whether there is
interdependence between the two types of responses, as occurs in septic
rats, despite attempts to shorten the duration of time for exposure to
insulin (29).
The E214k ubiquitin-carrier enzyme
is encoded by two species of mRNA, 1.8- and 1.2-kb mRNA. These mRNAs
differ in the length of their 3'-untranslated regions
(3'-UTR), but their translation products are identical (30). It
is not known whether these mRNAs are translated with equal efficiency.
Notably, Wing and Bedard (31) found that insulin-like growth factor I
or insulin will increase the rate of the 1.2-kb mRNA degradation but
not of the 1.8-kb species and concluded that the additional
3'-UTR in the 1.8-kb E214k
mRNA confers stability (31). This pattern of regulation of the
E214k mRNAs is interesting,
because we found that insulinopenia increases the 1.2- but not the
1.8-kb form. Our finding is consistent with activation of at least one
ubiquitin-carrier protein, E214k, and an increase in ubiquitin-conjugating activity in muscle
(unpublished observations). Taken together, these studies make it
tempting to speculate that one mechanism leading to suppression of
protein degradation by insulin could involve reduced activity of one or more ubiquitin-carrier enzymes. We do not know why the 1.8-kb E214k mRNA was increased in
muscles of diabetic rats given bicarbonate inasmuch as this response
was not found in any other group of diabetic rats. Possibly, the
increase in this mRNA was in response to bicarbonate directly or their
mild alkalosis (Table 1) rather than hyperglycemia or insulinopenia.
It is notable that the proteolytic response to acute diabetes and other
catabolic conditions (e.g., acidosis and sepsis) not only involves
glucocorticoids (17, 23, 28) but often is also associated with reduced
or impaired responses to insulin [e.g., uremia, sepsis, and
acidosis (7, 14, 17)], whereas starvation is associated with
reduced insulin levels. Thus impaired insulin action and/or relative
insulin deficiency could be a common factor activating protein
degradation in these conditions, since the present results show that a
low insulin level is a signal activating the ubiquitin-proteasome system.
In summary, muscle wasting in acute diabetes results from activation of
the ubiquitin-proteasome proteolytic pathway by a mechanism that
requires glucocorticoids. We have excluded a high blood glucose
concentration or acidification as independent mediators activating
muscle proteolysis in diabetes. It is tempting to speculate that the
insulin signal transduction pathway acts to suppress the
ubiquitin-proteasome system. In this case, insulin resistance in other
conditions (e.g., sepsis, burn injury, and metabolic acidosis) would
contribute to muscle atrophy, especially if glucocorticoid production
were high.
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ACKNOWLEDGEMENTS |
This study was supported by National Institutes of Health Grants
RO1-DK-37175, RO1-HL-45317, and RO1-DK-50740.
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FOOTNOTES |
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 and other correspondence: W. E. Mitch,
Renal Div., WMB 338, Emory University School of Medicine, 1639 Pierce
Dr., Atlanta, GA 30322 (E-mail: wmitch{at}emory.edu).
Received 23 November 1998; accepted in final form 11 February
1999.
 |
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