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Division of Cardiology, University Hospital of Geneva (M.B., J.C., A.A., P.D.), CH-1211 Geneva, Switzerland; and Renal Division, Emory University (S.R.P., J.L.B., W.E.M.), Atlanta, Georgia 30322
Address all correspondence and requests for reprints to: Dr. Marijke Brink, Division of Cardiology, Fondation pour Recherches Médicales, 64 avenue de la Roseraie, CH-1211 Geneva, Switzerland. E-mail: marijke.brink{at}dim.hcuge.ch
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We recently found that circulating IGF-I is markedly depressed in response to angiotensin II (ang II) infusion in the rat, concordant with marked weight loss (7). These responses are mediated by the AT1 receptor, but are independent of pressor responses to ang II. In this model 7- to 8-fold increases in ang II levels are obtained, similar to increases found in patients with congestive heart failure (CHF) (8, 9). CHF and chronic renal failure (CRF) are associated with weight loss, and an elevated circulating level of ang II could be a contributing factor (10, 11). Regarding mechanisms causing decreased lean body mass, muscle wasting in rats with CRF is caused by increased protein degradation via activation of the ubiquitin-proteasome system (12). The signals activating this system in CRF are complex, but IGF-I levels are low (4, 13), and as IGF-I influences both protein synthesis and degradation in muscle, a low circulating IGF-I level could account for reduced lean body mass when ang II levels are high. This led us to measure muscle protein turnover in response to ang II and manipulations of serum IGF-I levels. We found that the ang II-induced anorexia (7) is not the sole factor responsible for the decrease in muscle mass; there is also an increase in muscle protein degradation. Restoration of normal circulating IGF-I levels did not reverse the catabolic effects of ang II. We also found evidence for a marked depression of the autocrine skeletal muscle IGF-I system in rats infused with ang II. These findings could be important in understanding mechanisms of human disease, specifically the pathophysiology of muscle wasting in conditions such as CHF and CRF in which the renin-angiotensin system is activated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The body weight and food intake of each ang II-infused rat were measured daily, and a corresponding vehicle-infused control rat was given the same amount of food as that eaten by the ang II-infused rat on the previous day (i.e. pair-feeding). Three or 7 days after implantation of the osmotic pumps, rats were anesthetized, and aortic blood was withdrawn, mixed with EDTA in prechilled glass tubes, and immediately placed in ice. Plasma samples were stored at -80 C until IGF-I was measured by RIA. Tissues were removed, snap-frozen in liquid nitrogen, and stored at -80 C until processed.
Plasma IGF-I RIA and Western ligand blotting
Plasma samples were extracted with acid-ethanol to separate
IGF-binding proteins (IGFBPs) from IGF-I and assayed for IGF-I
immunoreactivity as previously described (14) using a
polyclonal anti-IGF-I rabbit antiserum provided by Drs. L. Underwood
and J. J. Van Wyk through the National Hormone and Pituitary
Program of the NIDDK, NIH. Standard curves were generated using human
recombinant IGF-I provided by Dr. H. P. Guler
(Ciba-Geigy, Summit, NJ). Western ligand blotting was
performed as previously described (7). Quantitative
analysis was performed by densitometry of autoradiograms.
Measurement of muscle protein turnover
The mixed fiber, epitrochlearis muscles were dissected from the
forelimbs of ang II-infused rats and pair-fed, vehicle-infused control
rats weighing about 200 g. To measure protein synthesis,
epitrochlearis muscles were preincubated for 30 min in Krebs-Ringer
bicarbonate, pH 7.4 (KRB), medium containing 10 mM glucose
and 0.5 mM L-phenylalanine. The muscles were
then blotted and placed in fresh KRB (3 ml) containing 0.5
mM L-phenylalanine and
L-[U-14C]phenylalanine (9.1 x
104 dpm/ml) (15, 16). After 2 h
of incubation, muscles were blotted and immediately frozen in liquid
nitrogen. Each muscle was homogenized in 10% trichloroacetic acid
using a ground glass homogenizer, and after centrifugation, the pellet
was washed twice with an ethanol-ether (1:1) mixture to remove
unincorporated
L-[U-14C]phenylalanine and then
dissolved in 0.3 N NaOH by incubation at 37 C.
Phenylalanine incorporated into protein was measured by liquid
scintillation counting to calculate the rate of protein synthesis. To
measure total protein degradation, epitrochlearis muscles were
preincubated at resting length for 30 min at 37 C in 95%
O2/5%CO2 in KRB containing
10 mM glucose (15, 16); cycloheximide (0.5
mM) was included to prevent reutilization of amino acids
released during protein breakdown to calculate the absolute rate of
protein degradation (16). The muscles were then placed in
fresh medium, regassed, and incubated for 2 h at 37 C.
Trichloroacetic acid (final concentration, 10%) was added to the
medium to remove peptides and proteins, and the amount of released
tyrosine was measured fluorometrically to calculate the rate of protein
degradation, because tyrosine is neither synthesized nor degraded in
muscle.
The influence of excessive circulating ang II on proteolytic pathways was evaluated by measuring the rates of protein degradation in epitrochlearis muscles incubated with inhibitors of different pathways while the contralateral muscle from the same rat was incubated without these inhibitors (12, 17, 18). Results were compared with those from muscles of the control rat incubated in the same fashion. Lysosomal proteolysis was inhibited by adding 1 mU/ml insulin, branched chain amino acids (200 µM valine, 170 µM leucine, and 100 µM isoleucine), and 10 mM methylamine, whereas calcium-activated proteases were inhibited by deleting calcium from the medium and adding 50 µM trans-epoxysuccinyl-L-leucylamido-(4-guanidino butane) (E-64), a potent inhibitor of the calpains and lysosomal proteases cathepsins B, D, H, and L (12, 17, 18). When we examined the contribution of the ubiquitin-proteasome pathway, the proteasome was inhibited by adding 30 µM MG132 to the medium plus inhibitors of the calpains and lysosomal proteases (12, 18, 19).
Northern blot analysis and solution hybridization/ribonuclease
(RNase) protection assay
Total RNA was prepared from frozen liver or gastrocnemius muscle
using Tri-Reagent (Molecular Research Center, Inc.,
Cincinnati, OH) and was assessed for purity by measuring absorptions at
260 and 280 nm. Total RNA (20 µg) was separated by electrophoresis in
a 1% agarose-formaldehyde gel, transferred to ZetaProbe GT
(Bio-Rad Laboratories, Inc., Hercules, CA) or Hybond C
(Amersham Pharmacia Biotech, Arlington Heights, IL)
membrane, and cross-linked to the membrane by UV irradiation. RNA
loading and transfer efficiencies were verified by methylene blue
staining of membranes or ethidium bromide staining of the gels. RNA was
hybridized with complementary DNA probes for ubiquitin,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), IGF-I
(20), IGFBP-3 (21), or IGFBP-5
(22) as previously described (12, 17, 18) or
using Quickhyb (Stratagene, La Jolla, CA) following the
manufacturer’s instructions. The amounts of hybridized probes for
ubiquitin, IGF-I, or the IGFBPs were quantified by PhosphorImager
(Molecular Dynamics, Inc., Sunnyvale, CA) and corrected
for variations in RNA using the corresponding GAPDH values.
IGF-I, IGF-I receptor (IGF-IR), and GAPDH messenger RNA (mRNA) levels in the gastrocnemius muscle were measured using solution hybridization/RNase protection assays as previously described (23).
Data analysis
All data represent the mean ± SEM of at least
six rats in each group, and results were analyzed using Student’s
t test when results from two experimental groups were
compared or ANOVA when data from three groups were studied. For data
analyzed by ANOVA, pairwise comparisons were made using Tukey’s test;
P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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]
consistent with our prior report (7). The decrease in
weight in controls was due to reduced food intake, but the
significantly stronger weight loss in ang II rats compared with
controls cannot be attributed to anorexia and decreased food intake,
because ang II and control rats were pair-fed (7). For the
rest of this study younger rats were used, because anorexia in younger
rats is less dramatic than that in older rats. Younger (
200 g) ang
II-infused rats gained significantly less weight than controls despite
identical food intake (12.9 ± 3.3 vs. 27.5 ±
3.0 g; P < 0.005; Fig. 1B). Reduced growth in
younger rats and weight loss in older rats have also been found when
responses to starvation were studied in rats of different ages
(24). Both suppression of growth and induction of weight
loss above the amount accounted for by variations in food intake are
consistent with a catabolic response to ang II.
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). Left ventricular and kidney
weights of ang II-infused rats were significantly higher than those in
controls, even when adjusted for body weight. Similar results occurred
when ang II-infused rats were compared with ad libitum-fed,
sham-infused rats (7). The weight of the epididymal fat
pad and its weight normalized to body weight [a measure of total body
fat content (25)] were not different after 7 days of ang
II infusion (Table 1). The weights of the predominantly red fiber
soleus, white fiber extensor digitorum longus, and mixed fiber
gastrocnemius muscles of ang II-infused rats were less than those of
the vehicle-infused, pair-fed, control rats (Table 1). The lower muscle
weight was not due to a change in water content, because we dried the
muscles and found that the dry weight of muscles was lower in ang II
rats than in pair-fed controls. Moreover, the hematocrit values in the
two groups were identical. Therefore, ang II must be inducing a
catabolic response in muscle.
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) was not eliminated by inhibiting
lysosomal and calcium-dependent proteases [i.e. blocking
these proteolytic systems reduced protein degradation to the same
degree (i.e. 16%) in muscles of control rats and ang
II-infused rats; P = NS]. These results indicate that
ang II stimulates a nonlysosomal and calcium-independent proteolytic
system. This response has generally been attributed to stimulation of
the ubiquitin-proteasome system (13). Consequently, we
evaluated the influence of this system by measuring protein degradation
when the proteasome inhibitor, MG132, was added. MG132 caused a sharp
decrease in protein breakdown in muscles from both groups. In some
experiments there was no difference in the rate of muscle proteolysis
between the groups, but when results from three experiments with five
rats per group in each experiment were combined, there remained a 13%
(P < 0.05) higher rate of muscle proteolysis in ang
II-infused rats. Moreover, we did not find a consistently increased
level of the mRNA encoding ubiquitin in muscles of ang II
vs. pair-fed control rats.
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The liver is the major source of circulating IGF-I, so we investigated
whether ang II infusion down-regulates hepatic IGF-I mRNA. Ang II
caused a significant 38% reduction in IGF-I mRNA (Fig. 3B) at 7 days vs. values in
pair-fed controls (P < 0.05; n = 6; Fig. 3C).
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) and 1.7-fold at 7 days compared
with levels in animals infused with ang II alone (P <
0.01 at both time points). Despite the increase in circulating IGF-I,
the body weight of rats coinfused with ang II and IGF-I was not greater
than that of rats infused only with ang II (Fig. 4A). Likewise, the lower gastrocnemius
weight (-0.14 ± 0.03 g) in ang II-infused rats (n =
13; P < 0.01) was not corrected by coinfusion of ang
II with IGF-I (n = 6; P = NS vs. ang II
alone; Fig. 4).
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showed that coinfusion of ang II and IGF-I significantly increased
IGFBP-3 (1.8-fold; n = 6; P < 0.05) compared with
levels in rats infused with ang II alone, thus resulting in levels that
were elevated even when compared with those in pair-fed control rats
(1.4-fold; n = 6; P = 0.07). Similar results were
obtained at 3 and 7 days. IGFBP-2 was increased in response to ang II
infusion (n = 6; P < 0.05). Coinfusion of IGF-I
and ang II resulted in IGFBP-2 levels that were 1.8-fold higher than
those in rats treated with ang II alone (n = 6; P
< 0.05), and 5.6-fold higher than those in pair-fed control rats
(n = 6; P < 0.05). Finally, IGFBP-4 was not
different between the ang II and ang II plus IGF-I groups (not shown).
Thus, IGF-I and ang II coinfusion leads to increased levels of
circulating IGF-I, IGFBP-3, and IGFBP-2 compared with ang II alone.
A potential mechanism for accelerated muscle protein breakdown is a
defect in the autocrine IGF-I system (4). Hence, we
analyzed the mRNAs for IGF-I, IGF-IR, and several IGFBPs in the
gastrocnemius muscle. Skeletal muscle IGF-I mRNA measured by a RNase
protection assay was 36% lower (P < 0.05
vs. control rats) after 7 days of ang II infusion (Fig. 5), and coinfusion of IGF-I and ang II
did not correct the decrease in IGF-I mRNA caused by ang II
(P < 0.05 vs. controls). IGF-IR mRNA in
gastrocnemius muscles was increased 20% after 7 days of ang II
(P < 0.01 vs. controls; Fig. 6), and coinfusion of ang II and IGF-I
resulted in a similar level of IGF-IR mRNA (P < 0.05
compared with controls; Fig. 7). Finally,
ang II decreased IGFBP-3 and IGFBP-5 mRNA levels in muscle compared
with controls (-60 ± 13% and -58 ± 10%;
P < 0.05 and P < 0.01, respectively).
Again, coinfusion of ang II with IGF-I did not correct the lower levels
of binding protein mRNAs. In all experiments we used GAPDH signals to
normalize for loading. GAPDH was not regulated by ang II or ang II plus
IGF-I. Our results indicate that ang II impairs the IGF-I autocrine
system in muscle.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Muscle protein synthesis was not significantly suppressed by ang II. However, when lysosomal and calcium-activated proteolytic pathways were blocked, protein degradation was high, indicating that ang II stimulates another proteolytic system in muscle. This usually implicates the ubiquitin-proteasome pathway (13), but we could not confirm this suspicion because of several confounding variables. Firstly, restricted food intake can stimulate the ubiquitin-proteasome system in muscle (30), and the anorexia induced by ang II infusion (7) superimposed on ang II-induced metabolic changes would increase the variability in measuring components of protein degradation and prevent us from identifying a specific pathway that is activated. Although anorexia was less severe in the younger rats that we used for this study, pair-fed control rats still had increased ubiquitin mRNA levels compared with ad libitum-fed controls (our unpublished observation). Secondly, there could be variability in the protein degradation measurements related to neurohormonal responses to ang II infusion (13). We and others found that glucocorticoids are required to stimulate the ubiquitin-proteasome pathway in acidosis, diabetes, and starvation (30, 31, 32), and we found that corticosterone excretion was high in some ang II rats, but the responses were quite variable (unpublished results). Thirdly, it is possible that ang II infusion stimulates another proteolytic pathway that has not been characterized (13).
Although IGF-I levels are regulated by dietary calories (6), we did not find any studies documenting that a chronically high ang II level suppresses IGF-I expression in both liver and skeletal muscle in vivo. On the other hand, we confirmed reports indicating that IGF-IR gene transcripts are increased by ang II (33). This is of interest because a similar combination of up-regulated skeletal muscle IGF-IR mRNA and down-regulated IGF-I mRNA levels has been described in rats with other conditions causing accelerated muscle proteolysis, including fasting, CRF, and diabetes (4, 6, 12, 18, 30, 34). However, the increase in IGF-IR expression was insufficient to overcome the higher rate of proteolysis even when the systemic IGF-I level was corrected by infusing IGF-I. This suggests that either the infused IGF-I was not bioavailable in the tissue or postreceptor IGF-I signaling may be impaired by ang II. Others have noted that ang II can induce abnormalities in the insulin signaling pathway, but whether this applies to IGF-I is unknown (35).
Modulation of IGFBP secretion can affect IGF-I-mediated muscle cell metabolism, proliferation, and differentiation (1, 3, 4, 5). Our data indicate that ang II decreases circulating IGFBP-3 and increases IGFBP-2. Coinfusion of ang II and IGF-I normalizes IGFBP-3 and further increases IGFBP-2. The increase in IGFBP-3 parallels the increase in IGF-I, suggesting that a larger pool of the IGFBP-3/IGF-I/acid-labile subunit complex circulates in these rats. From our data we cannot conclude whether the change in the IGFBP pattern results in a balance that prevents or augments the bioavailability of the infused IGF-I; however, our results in the heart of the same rats indicate that in tissues other then skeletal muscle IGF-I infusion can induce changes in muscle mass and tissue IGFBPs.
Our finding that IGF-I, IGFBP-3, and IGFBP-5 mRNAs are reduced in skeletal muscle tissue suggests that ang II impairs the autocrine IGF-I system and that correcting the circulating IGF-I level will not overcome this defect. There is a precedent for this conclusion, because Hsu et al. (36) demonstrated that skeletal muscle IGF-I gene expression and IGFBP-5 protein were decreased in potassium deficiency, another condition causing loss of muscle mass. Importantly, administration of IGF-I in that study did not result in muscle or body growth. They suggested that the autocrine changes in IGF-I and IGFBP-5 contribute to the attenuated muscle growth. Others have reported that overexpression of IGF-I in skeletal muscle is a potent myogenic stimulus (37), whereas viral-mediated expression of IGF-I blocked the aging-related loss of skeletal muscle function and maintained muscle mass and fiber types at levels similar to those in young adult rats (38). Transgenic mice with a liver-specific inactivation of the IGF-I gene have low circulating levels of IGF-I, but muscle growth is not impaired (39, 40). Taken together, these studies support the idea that a low serum level of IGF-I in response to ang II infusion is a less important stimulus for blunted skeletal muscle growth, but that decreased autocrine IGF-I responses contribute to muscle atrophy. It is also possible that a postreceptor defect contributes to the lack of the ability of circulating IGF-I to correct the reduction in muscle mass. However, this must be a tissue-specific defect, because in our studies there was an increase in cardiac mass in response to IGF-I infusion consistent with preserved IGF-IR signaling in cardiac tissue (23).
The differential effects of ang II in the heart, in which it increases
IGF-I, vs. other tissues such as skeletal muscle and liver,
in which it decreases IGF-I, are related to the fact that in the heart
it is a response to increased blood pressure (23), whereas
in the other tissues direct or indirect mechanisms may play a role,
including modulation of other hormone systems such as glucocorticoids,
testosterone, or tumor necrosis factor-
. We measured testosterone
concentrations; however, these were very variable and not statistically
different among the three groups (unpublished observation).
In summary, our findings could have implications for understanding the pathophysiology of conditions associated with chronic activation of the renin-angiotensin system and muscle wasting, such as CRF and CHF. In CRF, circulating IGF-I levels are low, but IGF-I administration has yielded disappointing results (41). In CHF, cachexia is a powerful predictor of outcome, and these patients have low IGF-I/GH ratios, suggesting some degree of GH resistance as has been found in CRF (42, 43). Notably, systemic administration of GH in CHF patients have yielded inconclusive results despite marked increases in circulating IGF-I. Our findings suggest that in these patients defects in the autocrine/paracrine IGF-I system could contribute to their cachexia.
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Footnotes |
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Received August 7, 2000.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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) was measured in one epitrochlearis muscles of ang
II-infused and pair-fed control rats, whereas the contralateral muscle
was incubated with inhibitors of lysosomal and calcium-dependent
proteases (
and 
).
Total proteolysis was increased by ang II (P <
0.05), and this response persisted after inhibiting lysosomal and
calcium-dependent proteolysis (P < 0.05). Shown
are the mean ± SEM of measurements from eight ang
II-infused and pair-fed control rats.
