INTRODUCTION

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ONE OF THE MOST PROMINENT metabolic changes seen during sepsis is the catabolic response in skeletal muscle, mainly caused by upregulated energy-ubiquitin-dependent protein breakdown, in particular myofibrillar protein breakdown (23, 24). During severe and protracted sepsis, continued muscle protein breakdown results in muscle wasting and fatigue, which may impair recovery and lead to an increased risk for thromboembolic and pulmonary complications if ambulation is delayed and respiratory muscles are affected. The ability to reduce the catabolic response in skeletal muscle during sepsis, therefore, would be of great clinical significance.

Previous studies provided evidence that insulin-like growth factor I (IGF-I) is a strong anabolic signal in muscle tissue. Treatment of cultured muscle cells with the hormone resulted in increased protein synthesis and inhibited protein breakdown (17, 21). In recent studies in our laboratory, IGF-I blocked the catabolic response in skeletal muscle following burn injury by stimulating protein synthesis and inhibiting protein breakdown (8).

In a study by Jurasinski and Vary (18), IGF-I increased protein synthesis in skeletal muscle of septic rats. The influence of the hormone on muscle protein breakdown during sepsis was not examined in that study, but such an effect may be even more important considering the significant role of stimulated muscle proteolysis in the catabolic response to sepsis. The purpose of the present study was to determine the effect of IGF-I on muscle protein breakdown during sepsis and to compare this effect with the hormonal regulation of protein synthesis in the same muscles.

	MATERIALS AND METHODS

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Experimental animals. Sepsis was induced by cecal ligation and puncture (CLP) in male Sprague-Dawley rats weighing 40-60 g as described previously (15, 20, 23). Control rats underwent sham operation, i.e., laparotomy and manipulation but no ligation or puncture of the cecum. All rats were resuscitated with 10 ml saline/100 g body wt administered subcutaneously on the back at the time of surgery. The rats had free access to water, but food was withheld after surgery to avoid any influence on metabolic changes of different food intake between the two groups of rats. This experimental model of sepsis is clinically relevant because it resembles the situation in patients with sepsis caused by intra-abdominal abscess and devitalized tissue. The model was characterized with respect to hemodynamic and metabolic changes and mortality rates (~30-40%) in previous studies from our (15, 20) and other laboratories (2). Rats weighing 40-60 g were used in the present experiments because lower extremity muscles from rats of this size are thin enough to allow for adequate tissue oxygenation and viability during in vitro incubation (12, 14). All experiments were conducted and animals were cared for in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Institutional Animal Care and Use Committee at the University of Cincinnati.

Muscle incubations. Protein turnover rates were measured in incubated extensor digitorum longus muscles 16 h after CLP or sham operation as described previously (13-15). The extensor digitorum longus muscle, which is a predominantly white fast-twitch muscle, was studied here because, in previous reports, sepsis-induced changes in protein metabolism were particularly pronounced in fast-twitch muscle (13, 15, 25). With rats under pentobarbital sodium anesthesia, the muscles were excised with intact tendons, mounted on stainless steel supports at resting length, and preincubated for 30 min at 37°C in 3 ml of oxygenated (95% O₂-5% CO₂) Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 10 mM glucose. The muscles were incubated at resting length, rather than flaccid, because in previous studies energy levels and metabolic rates were better maintained in muscles at resting length (1, 14).

For measurement of protein synthesis rate, muscles were transferred to 3 ml of fresh medium of the same composition as described above containing [U-¹⁴C]phenylalanine (0.5 mM; 0.05 µCi/ml). After incubation for 2 h, the amount of phenylalanine incorporated into trichloroacetic acid (10%)-precipitated proteins was determined as described in detail previously (13).

Total and myofibrillar protein breakdown rates were determined as net production of free tyrosine and 3-methylhistidine (3-MH), respectively, taking changes in tissue levels of the amino acids during incubation into account as described in detail previously (15, 23). After preincubation, one muscle was rinsed with fresh medium, blotted, weighed, and placed in ice-cold 3% (wt/vol) perchloric acid (PCA) for determination of tissue-free tyrosine and 3-MH. The contralateral muscle was transferred to fresh medium containing cycloheximide (0.5 mM) and incubated for 2 h. Cycloheximide was present in the medium to prevent reincorporation into protein of amino acids released during proteolysis. After incubation for 2 h, the muscle was rinsed, blotted, weighed, and placed in ice-cold 3% PCA. Muscles and media were stored at 20°C until tyrosine and 3-MH were assayed by high-performance liquid chromatography as described previously (15, 23).

To test the effect of IGF-I on muscle protein turnover rates, human recombinant IGF-I (kindly provided by Pharmacia, Stockholm, Sweden) was added to the medium at the start of the 2-h incubation period at concentrations ranging from 100 ng/ml to 10 µg/ml.

Statistics. Results are presented as means ± SE. Statistical comparisons were done by a two-way analysis of variance (sham vs. septic, IGF-I vs. no IGF-I) followed by Duncan's test.

	RESULTS

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Total and myofibrillar protein breakdown rates, measured as net release of tyrosine and 3-MH, respectively, were increased in muscles from septic rats (Fig. 1), similar to previous reports from our laboratory (15, 23). Treatment in vitro of the incubated muscles with 1 µg/ml of IGF-I inhibited both total and myofibrillar protein breakdown in muscles from sham-operated rats by ~25% but did not influence protein breakdown in muscles from septic rats (Fig. 1). The hormone concentration used in this experiment was based on results in a recent study in which protein breakdown was maximally inhibited by 1 µg/ml of IGF-I in muscles from nonburned and burned rats (8).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Total protein breakdown (tyrosine release) (A) and myofibrillar protein breakdown [3-methylhistidine (3-MH) release] (B) in muscles from sham-operated and septic rats incubated in the absence (open bars) or presence (filled bars) of 1 µg/ml of insulin-like growth factor I (IGF-I); n = 7 for each group. * P < 0.05 vs. no IGF-I; + P < 0.05 vs. corresponding sham group.

To study in greater detail the apparent resistance of protein breakdown to IGF-I in septic muscle, dose-response curves were established for muscles from sham-operated and septic rats with hormone concentrations ranging from 100 ng/ml to 1 µg/ml. Results from those experiments showed that protein degradation in muscles from sham-operated rats was inhibited in a dose-dependent manner, with a maximal effect noted at a concentration between 500 and 1,000 ng/ml (Fig. 2). In contrast, muscles from septic rats were completely resistant to the hormone with respect to its effect on total and myofibrillar protein breakdown (Fig. 2). The effects of the lower concentrations of IGF-I (100, 250, and 500 ng/ml) on protein breakdown rates in septic muscles were examined in this experiment despite the fact that the initial experiment indicated that protein breakdown in muscles from septic rats was resistant to 1 µg/ml of the hormone (see Fig. 1). This experimental design made it possible to perform a more complete comparison of the dose-response curves between nonseptic and septic muscles. In addition, it was important to rule out a biphasic response in muscles from septic rats with inhibition of protein breakdown only at lower hormone concentrations. In additional experiments, we tested the effects of higher concentrations (up to 10 µg/ml) of the hormone in septic muscle and, again, protein breakdown rates were not affected (Table 1). It should be noted that plasma levels of IGF-I in rats range from 100 to 200 ng/ml (5). Thus the higher hormone concentrations tested here are probably supraphysiological, although the relevance of the hormone concentrations in the in vitro system to in vivo hormone concentrations is not clear.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effect of different concentrations of IGF-I on total (A) and myofibrillar (B) protein breakdown in muscles from sham-operated () and septic rats (); n  7 for each data point. * P < 0.05 vs. no IGF-I.

Although the increase in protein breakdown is the most important factor in sepsis-induced muscle catabolism, at least from a quantitative standpoint, inhibited protein synthesis contributes to the metabolic response to sepsis (13, 25). We next examined the effect of IGF-I on muscle protein synthesis. When muscles were incubated in the presence of 1 µg/ml of IGF-I, protein synthesis was stimulated in both control and septic muscles (Fig. 3). The hormone did not completely block the sepsis-induced decrease in muscle protein synthesis, but protein synthesis in muscles from septic rats remained significantly lower than in muscles from sham-operated rats in the presence of 1 µg/ml of IGF-I (Fig. 3).

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Protein synthesis in muscles from sham-operated and septic rats with muscles incubated in the absence (open bars) or presence (filled bars) of 1 µg/ml of IGF-I; n = 8 for each group. * P < 0.05 vs. no IGF-I; + P < 0.05 vs. corresponding sham group.

To establish whether sensitivity or responsiveness to the hormone with regard to protein synthesis was altered during sepsis, muscles were incubated in the presence of different concentrations of IGF-I, ranging from 100 ng/ml to 1 µg/ml. Both when data were expressed in absolute values (Fig. 4A) and as percent increase in protein synthesis (Fig. 4B), results showed that sensitivity and responsiveness to the hormone of protein synthesis were not reduced in muscles from septic rats. In fact, protein synthesis was significantly increased by a lower concentration of IGF-I in muscles from septic than in muscles from sham-operated rats, suggesting that sepsis may be associated with an increased sensitivity of protein synthesis to the hormone.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effect of different concentrations of IGF-I on protein synthesis in muscles from sham-operated () and septic () rats. Results are given as nmol phenylalanine/g wet wt × 2 h (A) or percent increase in protein synthesis (B); n = 7 or 8 for each data point. * P < 0.05 vs. no IGF-I.

	DISCUSSION

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In the present study, addition of IGF-I to incubated muscles from nonseptic rats stimulated protein synthesis and inhibited protein breakdown in a dose-dependent manner, confirming previous reports of an anabolic effect of IGF-I in muscle tissue (8, 17, 18, 21). In contrast, the hormone did not influence protein breakdown in muscle from septic rats, not even when present at high concentrations, suggesting that protein breakdown in septic muscles becomes resistant to IGF-I. Because the response to the hormone of protein synthesis was not impaired in muscles from septic rats, the resistance of protein breakdown to the hormone most likely reflected a postreceptor defect.

A potential shortcoming of the present study was the use of small, growing rats, which could have influenced both the response to sepsis and the regulation by IGF-I of muscle protein metabolism. It should be noted, however, that in previous studies we found evidence that sepsis-induced changes in muscle protein turnover were similar in young and adult rats (27) and that the molecular regulation of muscle protein breakdown was similar in muscle tissue of adult septic patients (24) and young growing rats (23). Dardavet et al. (5) reported that old rats developed resistance to IGF-I in skeletal muscle secondary to a decrease in IGF-I receptors. Because IGF-I stimulated muscle protein synthesis to an even greater extent and at a lower hormone concentration in adult rats (18) than observed in the present study, it is not likely that the results reported here were only reflective of the young age of the rats.

A differential postreceptor regulation of protein synthesis and breakdown by IGF-I, as indicated by the results reported here, suggests that different intracellular pathways mediate the hormonal effects on protein synthesis and degradation. One of the earliest postreceptor events is phosphorylation of specific proteins that in turn activate various kinases. By using different kinase inhibitors, Dardavet et al. (6) found evidence that phosphatidylinositol (PI) 3-kinase is needed for the effects of IGF-I on both protein synthesis and degradation in skeletal muscle. The intracellular regulation by IGF-I of protein turnover downstream to PI 3-kinase, however, was different for protein synthesis and breakdown. Thus, whereas the stimulatory effect of IGF-I on protein synthesis was inhibited by the p70 S6 kinase blocker rapamycin, the inhibitory effect of IGF-I on protein breakdown was not influenced by rapamycin, supporting the concept of different signalling pathways for the regulation of protein synthesis and degradation by IGF-I in skeletal muscle.

The present result of IGF-I-stimulated protein synthesis in muscle from septic rats is similar to a recent report by Jurasinski and Vary (18). In that study, IGF-I was added to perfused rat hindlimbs and protein synthesis in the gastrocnemius muscle was stimulated in a dose-dependent manner with a maximal 2.5-fold increase seen at a hormone concentration of 1 nM (~ 7.5 ng/ml). One reason why muscle protein synthesis was stimulated at a lower IGF-I concentration in the study by Jurasinski and Vary (18) than in the current report may be that the hormone reached the tissue by the normal vasculature in the perfused hindlimbs as opposed to diffusion of the hormone into the incubated muscle tissue. Other explanations need to be considered as well, such as differences in production of IGF-I binding proteins between the two in vitro preparations. Although muscle protein breakdown becomes resistant to IGF-I during sepsis, the previous (18) and present findings of hormone-stimulated muscle protein synthesis are of clinical interest because blocking even one of the components of sepsis-induced muscle catabolism may improve muscle and whole body protein economy.

In addition to sepsis, the effect of IGF-I on muscle protein metabolism has been tested in other catabolic conditions as well. Ding et al. (7) found that muscles from rats with chronic renal failure were resistant to IGF-I with respect to both protein synthesis and degradation. In the same study, evidence was found for a postreceptor defect with impaired autophosphorylation of the IGF-I receptor beta subunit and decreased activity of the IGF-I receptor tyrosine kinase toward exogenous insulin receptor substrate-1. Impaired metabolic response to IGF-I in patients with chronic renal failure was reported by the same group (10).

In contrast to the findings in sepsis and chronic renal failure, burn injury, another condition characterized by pronounced muscle catabolism (9), does not seem to impair the response to IGF-I in skeletal muscle. In recent studies from our laboratory, treatment with IGF-I in vitro of muscles from burned rats stimulated protein synthesis and inhibited protein breakdown in a dose-dependent fashion, and the effect of the hormone on protein synthesis was even more pronounced in muscle from burned than from nonburned rats (8). In other experiments, we found that treatment of burned rats with IGF-I in vivo blunted the catabolic response in skeletal muscle (unpublished observations).

It is obvious, then, that different catabolic conditions may influence the responsiveness to IGF-I in skeletal muscle in different ways, with sepsis giving rise to hormone resistance of protein breakdown (present study), renal failure giving rise to resistance of both protein synthesis and degradation (7), and burn injury not impairing the response to IGF-I at all (8). The differential effects of various catabolic conditions on the responsiveness to IGF-I are important from a clinical standpoint because they suggest that treatment with IGF-I of patients with muscle catabolism needs to be tailored specifically to the underlying cause of the catabolic condition. The results may also explain why in some clinical studies treatment with IGF-I improved protein balance (3, 4), whereas in other studies the hormone had no beneficial effects (11, 19, 22). The mechanisms underlying the differential responsiveness to IGF-I in skeletal muscle in burn injury, sepsis, and other catabolic conditions as well are an important area for future research.

Although IGF-I may interact with both IGF-I and insulin receptors, there is evidence that in skeletal muscle, most of the metabolic effects of IGF-I at concentrations similar to those used in the present study are mediated by the IGF-I receptor (5). Interestingly, we previously found evidence that sepsis gives rise to a similar resistance to insulin as reported here for IGF-I, i.e., protein breakdown in muscle from septic rats became resistant to insulin, whereas protein synthesis responded to insulin in a normal way (16). Those results suggest that the insulin resistance in septic muscle reflected a postreceptor defect as well. It remains to be determined whether the resistance of protein breakdown in septic muscle to insulin and IGF-I is caused by the same intracellular mechanism.

In previous studies, we found that sepsis-induced muscle catabolism is caused mainly by upregulated ubiquitin-dependent proteolysis both in experimental animals (23) and patients (24). Other studies suggest that IGF-I may inhibit protein breakdown by reducing the expression of ubiquitin mRNA (8) and the levels of the 14-kDa ubiquitin-conjugating enzyme E2, secondary to increased breakdown of E2 mRNA (26). Thus it may be speculated that the resistance to IGF-I of protein breakdown in septic muscle may be caused by an impaired hormonal regulation of the ubiquitin-dependent proteolytic pathway, although further studies are needed to test that hypothesis.

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