INSULIN-LIKE GROWTH factor I (IGF-I) is an endocrine

Transcription

1 /01/$03.00/0 Vol. 142, No. 4 Endocrinology Printed in U.S.A. Copyright 2001 by The Endocrine Society Angiotensin II Induces Skeletal Muscle Wasting through Enhanced Protein Degradation and Down-Regulates Autocrine Insulin-Like Growth Factor I* MARIJKE BRINK, S. RUSS PRICE, JACQUELINE CHRAST, JAMES L. BAILEY, ASIF ANWAR, WILLIAM E. MITCH, AND PATRICK DELAFONTAINE 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 ABSTRACT We previously showed that angiotensin II (ang II) infusion in the rat produces cachexia and decreases circulating insulin-like growth factor I (IGF-I). The weight loss derives from an anorexigenic response and a catabolic effect of ang II. In these experiments we assessed potential catabolic mechanisms and the involvement of the IGF-I system in these responses to ang II. Ang II infusion caused a significant decrease in body weight compared with that of pair-fed control rats. Kidney and left ventricular weights were significantly increased by ang II, whereas fat tissue was unchanged. Skeletal muscle mass was significantly decreased in the ang II-infused rats, and a reduction in lean muscle mass was a major reason for their overall loss of body weight. In skeletal muscles, ang II did not significantly decrease protein synthesis, but overall protein breakdown INSULIN-LIKE GROWTH factor I (IGF-I) is an endocrine and autocrine/paracrine growth factor that has pleiotropic effects, including stimulation of cell growth and differentiation, erythropoiesis, chemotaxis, anabolism, and inotropy and prevention of apoptosis (1, 2). In skeletal muscle and cultured cells, IGF-I can increase the uptake of glucose and amino acids, enhance protein synthesis and suppress protein degradation (3, 4). Moreover, IGF-I stimulates mitogenesis in cultured skeletal muscle cells and induces myoblast differentiation (5). IGF-I is produced in many tissues, but the main source of circulating IGF-I is the liver, and the main regulators of hepatic IGF-I synthesis are GH and nutrient intake (6). 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 Received August 7, 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. marijke.brink@dim.hcuge.ch. * This work was supported by NIH Grants HL-47035, HL-45317, DK-45215, DK-37175, and DK-50740, the Swiss National Research Foundation (FNSR ), the Fonds Gerbex-Bourget, and the Swiss Cardiology Foundation. was accelerated; inhibiting lysosomal and calcium-activated proteases did not reduce the ang II-induced increase in muscle proteolysis. Circulating IGF-I levels were 33% lower in ang II rats vs. control rats, and this difference was reflected in lower IGF-I messenger RNA levels in the liver. Moreover, IGF-I, IGF-binding protein-3, and IGFbinding protein-5 messenger RNAs in the gastrocnemius were significantly reduced. To investigate whether the reduced circulating IGF-I accounts for the loss in muscle mass, we increased circulating IGF-I by coinfusing ang II and IGF-I, but this did not prevent muscle loss. Our data suggest that ang II causes a loss in skeletal muscle mass by enhancing protein degradation probably via its inhibitory effect on the autocrine IGF-I system. (Endocrinology 142: , 2001) 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. Animals Materials and Methods Male Sprague Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) were housed in individual metabolic cages. Osmotic minipumps (Alzet model 2001 or 1003D, Alza Corp., Palo Alto, CA) were implanted to infuse ang II at a rate of 500 ng/kg min or diluent (7); when indicated, IGF-I was infused at a rate of 1.4 mg/kg day. In a preliminary 1489

2 1490 METABOLIC ACTIONS OF ANGIOTENSIN II Endo 2001 Vol. 142 No. 4 experiment this dose resulted in circulating IGF-I levels similar to or greater than levels found in pair-fed control rats. All procedures were performed in accordance with institutional guidelines for the care of experimental animals. 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 IGFbinding 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- 14 C]phenylalanine ( dpm/ml) (15, 16). After2hofincubation, 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- 14 C]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% O 2 /5%CO 2 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 m valine, 170 m leucine, and 100 m isoleucine), and 10 mm methylamine, whereas calcium-activated proteases were inhibited by deleting calcium from the medium and adding 50 m transepoxysuccinyl-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 ubiquitinproteasome pathway, the proteasome was inhibited by adding 30 m 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 g) was separated by electrophoresis in a 1% agaroseformaldehyde 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. Results Body and organ weights When rats of approximately 300 g were infused with 500 ng/kg min ang II and compared with pair-fed, vehicleinfused, control rats, ang II caused a net weight loss of about 40 g over 7 days [ 81 7 vs g (control); P ; Fig. 1A] consistent with our prior report (7). The decrease in weight in controls was due to reduced food intake, but the FIG. 1. Ang II causes weight loss in older rats and limits growth in younger rats. Body weight was determined in 8- to 9-week-old rats (A) or 6-week-old rats (B) before implantation of osmotic minipumps (Initial) and after 6 days (Final) of infusion of pair-fed control and ang II-infused rats. Shown are the mean SEM of measurements from six or seven rats.

3 METABOLIC ACTIONS OF ANGIOTENSIN II 1491 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 ( vs 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. We weighed the organs and hindlimb muscles from ang II-infused and pair-fed control rats 3 or 7 days after beginning the ang II infusion (Table 1). 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. Protein synthesis and degradation To examine pathways that could account for the imbalance in protein synthesis and degradation, we measured these rates in the mixed fiber epitrochlearis muscle of ang IIinfused and pair-fed control rats weighing about 200 g. This muscle was studied because protein synthesis and degradation measurements in it respond in the same fashion as those in the bulk of muscle in the rat (16) and are similar to responses measured in vivo in awake rats (17, 26). A small part of the difference in muscle mass may have been due to lower levels of protein synthesis, as synthesis in muscles of ang II rats ( nmol phenylalanine/g h) was somewhat lower than that in muscles of control rats ( nmol/ g h); however, the difference was not statistically significant (P 0.09). Consequently, the lower muscle mass must be due to excessive protein degradation. First, we tested whether lysosomal and calcium-activated proteases are increased in response to ang II by incubating one epitrochlearis muscle under basal conditions and the contralateral muscle under conditions that inhibit lysosomal function and calcium-activated proteases (12, 17, 18). The basal rate of protein degradation was higher in muscles of ang II-infused rats (198 9 nmol tyrosine/g h) than in control rats (159 9 nmol tyrosine/g h (P 0.05), and this difference (Fig. 2) 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 calciumindependent 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. Circulating IGF-I and liver IGF-I mrna Previously, we found that plasma IGF-I levels are sharply reduced in ang II-infused vs. sham-infused control rats fed ad libitum (7). In the present study plasma IGF-I levels in ang II-infused rats were reduced by 33% after 3 days (P 0.01) and by 26% after 7 days of treatment (P 0.05) vs. values in pair-fed controls. 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). Responses to Ang II and IGF-I coinfusion We examined whether the lower circulating level of IGF-I is involved in the accelerated rate of muscle protein breakdown by infusing rats with ang II or both ang II and IGF-I. The ang II and IGF-I combination increased circulating IGF-I TABLE 1. Organ weights of tissues from Ang II and pair-fed control rats Day Left ventricle (g) Kidney (g) Epididymal fat (g) Epididymal fat/bw ratio (mg/g) Soleus (mg) EDL (mg) Gastrocnemius (g) 3 Control ND ND ND 3 Ang II a a b ND ND ND 7 Control ND Ang II b ND a a a Values are the mean SEM of measurements made at 3 days (n 6) or 7 days (n 6 7) of infusion. ND, Not determined. a P 0.01 vs. control. b P 0.05.

4 1492 METABOLIC ACTIONS OF ANGIOTENSIN II Endo 2001 Vol. 142 No fold at 3 days (Table 2) 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 ( 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). As tissue availability of IGF-I is determined by the balance between IGF-I and its specific binding proteins, we analyzed circulating IGFBPs by Western ligand blotting. Compared with levels in pair-fed controls, ang II decreased IGFBP-3 at 3 and 7 days of infusion, consistent with our previous report. Quantitative analysis of autoradiograms (Table 2) 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. FIG. 2. Ang II infusion stimulates muscle protein degradation. After 7 days of ang II or vehicle infusion, total protein degradation ( ) 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 f). 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. Discussion A major finding in our study is that the failure of normal growth of young rats induced by ang II is due to impaired accumulation of muscle protein, the major fraction of lean body mass (27, 28). In older rats that lost weight vs. pair-fed, control rats, the hematocrit values were not different, indicating that the weight loss was not due to dehydration. Changes in epididymal fat pad weight are closely related to changes in total fat mass (25), so our finding that fat pad mass is maintained during ang II infusion is consistent with impaired muscle protein turnover rather than fat loss as the mechanism for failure to gain weight. Others report that ang II can cause a loss of retroperitoneal fat (29), but as we did not measure this specific organ, it is possible that some loss of this tissue contributed minimally to the weight loss. We did find increased muscle protein breakdown in response to ang II, which would contribute to weight loss. The mechanism for this proteolytic response could be related to changes in the IGF-I system. There were low levels of circulating IGF-I, but correcting this level did not lead to normal growth, whereas there was evidence for impairment of the autocrine IGF-I system in skeletal muscle, including significantly reduced levels of expression of IGF-I, IGFBP-3, and IGFBP-5 mrnas. Finally, the response of skeletal muscle to ang II contrasts with that of cardiac muscle, because there was hypertrophy, and the level of IGF-I is increased by ang II (23). It is important to note that the IGF-I we infused was bioactive, because IGFBP-4 and IGFBP-5 mrna levels were higher in the hearts of rats coinfused with ang II and IGF-I than in rats infused with ang II alone (unpublished data). Taken together, these results show that correction of systemic IGF-I is ineffective in preventing the ang II-induced loss in skeletal muscle mass in our model and suggest that the autocrine IGF-I system plays a role in determining muscle protein mass. 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 ubiquitinproteasome 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 iden-

5 METABOLIC ACTIONS OF ANGIOTENSIN II 1493 FIG. 3. Systemic IGF-I and liver IGF-I mrna levels in ang II-infused and pair-fed control rats. Total circulating IGF-I levels in plasma at 3 and 7 days of infusion were measured by RIA as described in Materials and Methods (A). B, Northern blot autoradiogram assessing the abundance of mrna encoding IGF-I (top panel) in the liver of two representative pairs, each pair consisting of an ang II-infused rat and a pair-fed control rat, at 7 days of infusion. GAPDH (bottom panel) was used as a loading control. C, The abundance of IGF-I mrna was quantitated after correction for GAPDH mrna abundance. Shown are the mean SEM of determinations from six rats at 7 days of infusion. TABLE 2. Circulating IGF-I and IGFBP levels of Ang II, Ang II plus IGF-I, and pair-fed control rats IGF-I ( g/liter) IGFBP-3 (Dens. units) IGFBP-2 (Dens. units) Control Ang II a b b Ang II IGF b,c d b,d Values are the mean SEM (n 6). Dens. units, Densitometry units. a P 0.01 vs. control. b P 0.05 vs. control. c P 0.01 vs. ang II. d P 0.05 vs. ang II. tifying 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 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 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

6 1494 METABOLIC ACTIONS OF ANGIOTENSIN II Endo 2001 Vol. 142 No. 4 FIG. 4. IGF-I coinfusion with ang II does not prevent decreases in body mass and gastrocnemius muscle mass. A, Changes in body weight were determined in 6-week-old rats before implantation of osmotic minipumps (Initial) and after 6 days (Final) of infusion (the day before sacrifice) of pair-fed control, ang II-infused, and IGF-I/ang II-coinfused rats. Weights are expressed as a percentage of body weight at the start of ang II infusion. B, After 7 days the gastrocnemius muscles of the same three groups of rats were dissected and weighed. Shown are the mean SEM of measurements from six or seven rats per experimental group. FIG. 5. Gastrocnemius muscle IGF-I mrna levels in ang II-infused, pair-fed control and IGF-I/ang II-coinfused rats. A, Representative autoradiogram of solution hybridization analysis to assess abundance of mrna encoding IGF-I in the gastrocnemius of the three groups of rats at 7 days of infusion. B, The abundance of IGF-I mrna was quantitated after correction for GAPDH mrna abundance. Shown are the mean SEM of determinations from six rats per experimental group. Ang II decreases IGF-I mrna in gastrocnemius muscle (P 0.05 vs. control), and IGF-I coinfusion with ang II did not prevent this decrease (P 0.05 vs. control). 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). FIG. 6. Gastrocnemius muscle IGF-IR mrna levels in ang IIinfused, pair-fed control and IGF-I/ang II-coinfused rats. A, Representative autoradiogram of solution hybridization analysis to assess abundance of mrna encoding IGF-IR in the gastrocnemius of the three groups of rats at 7 days of infusion. B, The abundance of IGF-IR mrna was quantitated after correction for GAPDH mrna abundance. Shown are the mean SEM of determinations from six rats per experimental group. In rats infused with ang II or coinfused with ang II/IGF-I, IGF-IR mrna in gastrocnemius muscle was increased (P 0.05 vs. control). 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),

7 METABOLIC ACTIONS OF ANGIOTENSIN II 1495 FIG. 7. Gastrocnemius muscle IGFbinding protein-3 (IGFBP-3) and IGF- BP-5 mrna levels in ang II-infused, pair-fed control rats and IGF-I/ang II-coinfused rats. A, Representative Northern blot autoradiogram to assess the abundance of the mrnas encoding IGFBP-3 and IGFBP-5 in the gastrocnemius of ang II-infused, ang II/IGF-I-coinfused, and pair-fed control rats at 7 days of infusion. B, The abundance of IGFBP-3 and IGFBP-5 mrna was quantitated after correction for GAPDH mrna abundance. Shown are the mean SEM of determinations from six rats per experimental group. IGFBP-3 and IGFBP-5 mrna levels in gastrocnemius muscle were decreased (P 0.05 vs. control) in rats infused with ang II or coinfused with ang II/IGF-I. 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. References 1. 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