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Department of Surgery, University of Cincinnati, and Shriners Hospital for Children, Cincinnati, Ohio 45267-0558
Address all correspondence and requests for reprints to: Per-Olof Hasselgren, M.D., Department of Surgery, University of Cincinnati, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0558. E-mail: hasselp{at}uc.edu
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Abstract |
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Introduction |
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Previous studies suggest that insulin-like growth factor I (IGF-I) exerts an anabolic effect in muscle tissue. For example, treatment of cultured muscle cells with the hormone resulted in increased protein synthesis and reduced protein degradation (9, 10). In recent studies in our laboratory, IGF-I blocked the catabolic response in skeletal muscle after burn injury, both when incubated muscles were treated in vitro (11) and when burned rats were treated with the hormone in vivo (12).
In contrast, when incubated muscles from septic rats were treated with IGF-I, protein degradation was unaffected, even at high hormone concentrations, suggesting that muscle becomes resistant to IGF-I during sepsis (13). Because the muscles were treated with the hormone in vitro in those experiments and because the metabolic and hormonal milieu is much more complex in vivo than in vitro, the results observed in vitro do not necessarily mean that muscle becomes resistant to treatment with IGF-I in vivo during sepsis. In the present study, we tested the hypothesis that administration of IGF-I in vivo stimulates protein synthesis and inhibits protein breakdown in skeletal muscle during sepsis. Because, in several previous reports, sepsis-induced muscle proteolysis was associated with increased gene expression of ubiquitin (6, 7) and other components of the ubiquitin-proteasome proteolytic pathway, including the ubiquitin-conjugating enzyme E214k (5), messenger RNA (mRNA) levels for ubiquitin and E214k were determined, as well.
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Materials and Methods |
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Immediately after the sc implantation of the miniosmotic pumps, groups of rats underwent induction of sepsis or sham-operation. Sepsis was induced by cecal ligation and puncture (CLP), as described previously (4, 5, 7, 13). Sham-operated rats underwent laparotomy and manipulation but no ligation or puncture of the cecum. All rats were resuscitated with saline (10 ml/100 g BW), administered sc on the back, at the time of surgery. The rats were randomly assigned to one of four groups: 1) sham-operation + saline; 2) sham-operation + IGF-I; 3) CLP + saline; or 4) CLP + IGF-I. After the procedures, the rats were housed individually and were allowed free access to drinking water. Food was withheld after surgery to avoid any influence on metabolic changes of different food intake between the groups of rats. Metabolic studies were performed 16 h after CLP or sham-operation. In previous studies, rats were in a hyperdynamic phase of sepsis (14, 15), and ubiquitin-proteasome-dependent muscle proteolysis was increased 16 h after CLP (7). The experimental model of sepsis used here is clinically relevant because it resembles the situation in patients with sepsis caused by intraabdominal abscess and devitalized tissue. The model was characterized in previous reports from our (15) and other laboratories (14), with regard to mortality rates and hemodynamic and metabolic changes. Rats weighing 50–60 g were used because lower extremity muscles from rats of this size are thin enough to allow for adequate tissue oxygenation and viability during in vitro incubation (2, 16). 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
Sixteen hours after CLP or sham-operation, rats were
anesthetized with pentobarbital (35 mg/kg ip). The extensor digitorum
longus (EDL) muscles were dissected with intact tendons and mounted on
stainless steel supports at resting length and preincubated for 30 min
at 37 C in 3 ml oxygenated
(O2:CO2 = 95:5)
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
(16, 17).
For measurement of protein synthesis rate, muscles were transferred to 3 ml 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 previously (11, 13).
For measurement of protein breakdown rates, muscles were preincubated in oxygenated medium for 30 min, as described above. After preincubation, one muscle was rinsed with fresh medium, blotted, weighed, and placed in ice-cold 0.4 N perchloric acid for determination of tissue-free tyrosine and 3-methylhistidine (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 0.4 N perchloric acid. Muscles and media were stored at -20 C until tyrosine and 3-MH were assayed by HPLC. Total and myofibrillar protein breakdown rates were determined as net release of tyrosine and 3-MH, respectively, taking changes in tissue levels of the amino acids during incubation into account, as previously described in detail (2, 16).
Northern blot analysis
Sixteen hours after CLP or sham-operation, rats were
anesthetized with pentobarbital (35 mg/kg ip). The extensor digitorum
longus muscles were harvested, immediately frozen in liquid nitrogen,
and then stored at -70 C until analysis. Messenger RNA levels for
ubiquitin and the 14-kDa ubiquitin-conjugating enzyme
E214k were determined by Northern blot analysis,
as previously described in detail, using
32P-labeled cDNA probes (5, 6). Blots were
quantitated on a Phosphoimager using the Image Quant Program
(Molecular Dynamics, Inc., Sunnyvale, CA), and the
relative mRNA abundance was determined by using a rat 18S ribosomal
probe to control for equal loading of the lanes.
Plasma glucose and amino acids
Blood was collected, by heart puncture, at the time of muscle
dissection. Plasma glucose was determined by a colorimetric assay using
Vitros GLU slides (Johnson & Johnson Clinical Diagnostics,
Rochester, NY). Plasma amino acids were measured in an amino acid
analyzer (Beckman Coulter, Inc. 6300; Beckman Coulter, Inc., Palo Alto, CA).
Statistics
Results are presented as mean ± SEM.
Statistical comparisons were done by Student’s t test or
ANOVA followed by Duncan’s test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). There was no
mortality among sham-operated rats treated with saline or IGF-I.
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). Administration of IGF-I did not
influence muscle protein synthesis rates in sham-operated rats.
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), which is in line with previous
reports from this laboratory (2, 4, 7, 13). Total muscle protein
breakdown was not influenced by treatment with IGF-I in septic or
sham-operated rats. Similar results were observed in three repeated
experiments.
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).
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).
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). The sizes of the ubiquitin
transcripts noted here (2.6 and 1.2 kb) are similar to previous reports
(6, 18, 19). Sepsis influenced the expression of both these transcripts
to a similar degree, and both were therefore included in the
quantitation. A smaller ubiquitin transcript (approximately 0.5 kb) was
also present but was not influenced by sepsis (not shown). Because in
several previous reports, parallel changes in muscle protein breakdown
rates and ubiquitin mRNA levels occurred, we did not expect ubiquitin
mRNA to be influenced by IGF-I treatment in the present experiments.
Surprisingly, however, ubiquitin mRNA levels were normalized in muscle
from septic rats treated with IGF-I (Fig. 4). Identical results were
observed in three repeated experiments.
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). A similar
selective increase in the concentration of the small
E214k transcript was observed during sepsis, in a
recent study from our laboratory (5), and was reported in other
catabolic conditions, as well (20, 21). Treatment with IGF-I blocked
the sepsis-induced increase in muscle E214k mRNA
levels (Fig. 5).
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Although changes in plasma amino acids are a rather nonspecific
indication of changes in whole-body protein metabolism, changes in the
plasma concentrations of certain amino acids (including phenylalanine,
tyrosine, histidine, and the branched chain amino acids) have been
considered to reflect changes in muscle protein breakdown rates
(22, 23, 24). Sepsis resulted in increased plasma concentrations of most of
these amino acids, and most of the sepsis-induced changes in plasma
amino acids were reversed by treatment with IGF-I (Table 3). Indeed, the majority of plasma amino
acids were reduced during treatment with IGF-I, both in sham-operated
and septic rats, and total amino acids were reduced by the hormone in
both groups of rats.
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).
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). We next measured muscle protein breakdown rates after
administration of 1.2 g glucose to septic rats receiving IGF-I.
Also, in this experiment, muscle protein breakdown rates were not
influenced by the hormone (Fig. 6). The
lower protein breakdown rates noted in septic rats in this, rather than
our first experiment (compare with protein breakdown rates in septic
rats in Fig. 2), may reflect an inhibitory effect of glucose on muscle
protein degradation.
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Discussion |
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A differential postreceptor regulation of protein synthesis and degradation by IGF-I, as indicated by the present and a recent study (13), is consistent with the concept that different intracellular signaling pathways mediate the hormonal effects on muscle protein synthesis and breakdown. A similar conclusion was reached in a previous report in which we found that the stimulation of protein synthesis by insulin was not affected by sepsis, whereas inhibition of protein breakdown in septic muscle required higher concentrations of insulin than in nonseptic muscle (2, 29). The results in the present study and in a recent in vitro study (13) suggest that muscles become completely unresponsive to IGF-I during sepsis. This differs from other experiments in which insulin inhibited protein breakdown in septic muscle, although to a much smaller degree than in nonseptic muscle (2, 29). Thus, the degree of resistance to IGF-I and insulin in septic muscle may differ between the two hormones. The mechanism of the sepsis-induced resistance in skeletal muscle to IGF-I is not known from the present study, and it is not known whether the same mechanism accounts for the postreceptor IGF-I and insulin resistance. It should be noted that insulin resistance and IGF-I resistance do not always occur simultaneously. For example, muscle glucose uptake during sepsis is resistant to insulin but not to IGF-I (28), suggesting that different mechanisms account for the resistance to the two hormones.
The present finding, that administration of IGF-I to sham-operated rats did not stimulate muscle protein synthesis or inhibit protein breakdown, was surprising and is in conflict with several previous studies in rats. It should be noted, however, that in most previous experiments in which IGF-I stimulated basal muscle protein synthesis (including using studies from our laboratory), the tissue was treated with the hormone in vitro (11, 13, 30, 31). Where rats were infused in vivo with IGF-I, using an identical protocol as used here, basal muscle protein synthesis rates were not affected (12). In contrast, acute iv administration of IGF-I (60 min) in rats resulted in increased muscle protein synthesis determined by a flooding dose of phenylalanine (32). Although plasma amino acids were not measured in that study, it may be speculated that the treatment period was too short to result in hypoaminoacidemia, whereas in the present and in our previous report (12), infusion of the hormone reduced plasma amino acids. A recent study by Fryburg et al. (33) suggests that the anabolic effects of IGF-I may be prevented by hypoaminoacidemia.
Another reason why the present and some of the previous reports, with regard to the effect of IGF-I on basal muscle protein turnover rates, are apparently conflicting may be the potential role of IGF-I binding proteins (34). Although binding proteins may be produced by muscles in vitro, it is possible that long-term administration of IGF-I in vivo results in greater changes in IGF-I binding proteins than short-term treatment in vivo or in vitro.
It may be argued that the lack of effect of IGF-I on muscle protein breakdown noted here in septic rats reflected an insufficient amount of the hormone administered, rather than hormone resistance. Though this may be true, it should be noted that an identical regimen of hormone administration as that used in the present study blocked the burn-induced increase in muscle proteolysis in a recent study from our laboratory (12). In the same study, total plasma IGF-I levels were increased from 386 to 504 ng/ml in burned rats treated with the hormone. Interpretation of plasma IGF-I levels, under the present experimental conditions, is complicated by the finding that rat plasma IGF-I levels are reduced when human IGF-I is administered to rats (12). In addition, changes in IGF-I binding proteins may influence the bioavailability of the hormone. It should be noted that when muscles were treated with IGF-I in vitro, protein degradation was not inhibited by the hormone in septic muscle, even at hormone concentrations approximately five times normal plasma concentrations (13), further supporting the concept that the lack of effect of IGF-I on muscle proteolysis during sepsis does not reflect an insufficient amount of the hormone. More important, the hypoglycemia that is associated with administration of IGF-I is a limiting factor (25). Severe hypoglycemia developed with the rate of IGF-I administration used in the present experiments and administration of an even larger amount of the hormone would probably not be clinically relevant.
Because hypoglycemia stimulates the secretion of glucagon and epinephrine (26, 27), it is possible that hypoglycemia-associated hormonal changes offset the anabolic effects of IGF-I. However, the present observation that IGF-I did not reduce muscle protein breakdown in septic rats, even after supplementation with a large amount of glucose, suggests that the lack of effect of IGF-I on muscle proteolysis was not caused by hypoglycemia alone.
In addition to sepsis, the effect of IGF-I on muscle protein metabolism has been tested in other catabolic conditions, as well. In an experimental model of chronic renal failure, muscles were resistant to IGF-I, with respect to both protein synthesis and degradation (35). In the same report, evidence was found for impaired autophosphorylation of the IGF-I receptor ß-subunit and decreased activity of the IGF-I receptor tyrosine kinase toward insulin receptor substrate-1. The same group reported impaired metabolic response to IGF-I in patients with chronic renal failure (36).
In contrast to the situation in sepsis and chronic renal failure, burn injury [another condition characterized by muscle cachexia (37, 38)] is not associated with resistance to IGF-I. Thus, in recent studies, we found that treatment of burned rats in vivo (12) or of muscles from burned rats in vitro (11) with IGF-I stimulated protein synthesis and inhibited protein breakdown in a dose-dependent fashion and the effect of the hormone on protein synthesis were even more pronounced in muscle from burned, than from nonburned, rats.
It is obvious, then, that different catabolic conditions may influence the responsiveness to IGF-I in skeletal muscle differentially, with sepsis giving rise to hormone resistance of protein degradation (Ref. 13 and present study), renal failure resulting in resistance of both protein synthesis and breakdown (35), and burn injury not giving rise to hormone resistance at all (11, 12). These observations suggest that treatment with IGF-I of patients with muscle cachexia needs to be tailored specifically to the cause of the catabolic condition. The results may also explain why, in some clinical studies, administration of IGF-I improved protein balance (39, 40); whereas, in other studies, the hormone had no beneficial effect (41, 42).
The reduced mRNA levels for ubiquitin and the ubiquitin-conjugating enzyme E214k, in muscles of septic rats treated with IGF-I, were surprising, in light of the unchanged protein breakdown rates in the same muscles. In several previous studies, muscle protein breakdown rates and mRNA levels for ubiquitin and other components of the ubiquitin-proteasome proteolytic pathway were up- or down-regulated in parallel (43). It should be noted, however, that there is not an absolute correlation between steady-state levels of mRNA for proteolytic enzymes or other components of proteolytic pathways and the actual proteolytic activity in that pathway (44). The present observations of unchanged protein breakdown rates and reduced mRNA levels for ubiquitin and E214k in muscles from septic hormone-treated rats, therefore, are not necessarily contradictory. The mechanism(s) of reduced mRNA levels for ubiquitin and E214k in hormone-treated septic rats is not known from the present study, but the results may reflect increased breakdown of mRNA (45), in addition to inhibited gene transcription. Regardless of the mechanism, the observations are important because they suggest that changes in mRNA levels for ubiquitin and E214k do not always reflect changes in muscle protein breakdown rates.
It may be argued that comparisons between protein breakdown rates and mRNA levels cannot be done under the present experimental conditions because protein breakdown rates were determined in muscles incubated for a total of 2.5 h (30 min preincubation and 2 h incubation), whereas mRNA levels were measured in muscles that were frozen immediately after dissection. It should be noted, however, that although muscles were incubated for a total of 2.5 h, the protein turnover rates did not reflect the metabolic activity only at the end of incubation but reflected the protein synthesis and breakdown rates during the 2-h incubation period. With the present in vitro technique, protein turnover rates are constant during incubation for at least 2 h in most conditions (16). Thus, the protein breakdown rate is basically the same at the start and end of incubation, and comparisons between protein breakdown rates (determined with the present in vitro technique) and mRNA levels for ubiquitin and E214k, therefore, are valid.
Changes in plasma amino acids have been frequently used as an indicator of changes in muscle protein breakdown rates during sepsis and other catabolic conditions (22, 23, 24). The present results, however, strongly suggest that changes in plasma amino acids do not only reflect changes in muscle proteolysis, at least not under the present experimental conditions. Although it may be argued that the changes in plasma amino acids reflected protein turnover rates in muscles other than the extensor digitorum longus muscle, this is less likely because we have found previously that sepsis mainly induces a catabolic response in white, fast-twitch skeletal muscle (e.g. extensor digitorum longus muscle), with only minor changes noted in other types of muscle (2, 6).
Changes in plasma amino acids may be caused by a number of different factors in addition to changes in muscle protein turnover rates, such as changes in metabolism of the individual amino acids, tissue uptake or release or urinary excretion of amino acids, and changes in protein or amino acid metabolism in organs and tissues other than skeletal muscle. Despite the fact that changes in plasma amino acids may be rather nonspecific, the present observations are important because they suggest that the amount of hormone administered was sufficient to block some of the sepsis-induced changes in protein or amino acid metabolism, resulting in changes in plasma amino acid levels. Further studies are needed to define which metabolic alteration(s) caused the changes in plasma amino acids noted here.
In summary, the present results suggest that administration of IGF-I may improve sepsis-induced muscle cachexia by stimulating protein synthesis. However, because muscles were resistant to IGF-I, with regard to the regulation of protein breakdown, the use of IGF-I to treat muscle cachexia during sepsis remains unclear. Because at least some of the mechanisms of sepsis-induced muscle proteolysis are similar in rats and humans (7), it is possible that muscle proteolysis becomes resistant to IGF-I in septic patients also, although further studies are needed to test that notion. It will be important, in the future, to determine the intracellular mechanisms of the unresponsiveness to IGF-I in skeletal muscle during sepsis.
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Footnotes |
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Received November 29, 1999.
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7 in each group. *, P < 0.05
vs. all other groups by ANOVA.
