Evidence That Tumor Necrosis Factor Participates in the Regulation of Muscle Proteolysis During Sepsis Oded Zamir, MD; Per-Olof Hasselgren, MD, PhD; Steven L. Kunkel, PhD; Janice Frederick, MD; Takashi Higashiguchi, MD; Josef E. Fischer, MD
role of tumor necrosis factor (TNF) in the regulation of muscle protein turnover was studied in rats. Protein synthesis and total and myofibrillar protein breakdown rates were measured in incubated extensor digitorum longus muscles. Intraperitoneal administration of recombinant TNF-\g=a\(300 \g=m\g/kgof body weight) increased total and myofibrillar protein breakdown rates by 28% and threefold, respectively, with no effect on protein synthesis. In subsequent experiments, sepsis was induced by cecal ligation and puncture or a sham-operation was performed. Rats received TNF antiserum (1 mL/100 g of body weight) or control serum 2 hours before cecal ligation and puncture or shamoperation. Treatment with TNF antiserum reduced the mortality rate from 25% to 5% following cecal ligation and puncture. The treatment had no effect on protein synthesis but reduced total and myofibrillar protein breakdown rates by 26% and 39%, respectively, in septic animals. Results suggest TNF is involved in the regulation of sepsis-induced muscle proteolysis.
metabolism when added to incubated when administered in vivo.9 In contrast, other reports suggest that TNF can induce muscle catabolism,10"12 possibly through stimulation of or interac¬ tion with adrenocortical hormones.1314 Furthermore, even if TNF can stimulate muscle protein breakdown when administered to nonseptic subjects, it does not necessar¬ ily mean that TNF actually participates in the regulation of muscle proteolysis during sepsis. The purpose of the present study was twofold. First, we tested the hypothesis that administration of TNF to nor¬ mal rats results in a sepsislike metabolic response in skel¬ etal muscle, that is an increased breakdown of predomi¬ nantly myofibrillar proteins. Second, we tested the hypothesis that TNF plays a role in the regulation of muscle proteolysis during sepsis by measuring muscle protein turnover rates in septic rats that had been treated with antiserum against TNF.
catabolism is a characteristic metabolic re¬ sponse to sepsis that mainly reflects increased pro¬ tein breakdown, especially myofibrillar protein break¬ down.1-2 Although this response may be beneficial to the organism during the early phase of sepsis by providing the liver with increased amounts of amino acids for
Sprague-Dawley rats (Zivic-Miller, Zelienople, Pa) weighing from 40 to 60 g were used. Rats of this size possess ex¬ tensor digitorum longus muscles that are thin enough to allow adequate diffusion of oxygen and substrates into tissue during incubation.15 The animals were maintained on regular rat chow,
\s=b\ The
to affect
(Arch Surg. 1992;127:170-174)
Muscle
acute-phase protein synthesis,3 a protracted degradation of muscle proteins will result in muscle wasting, fatigue, and delayed recovery. Recent studies suggest that both glucocorticoids and different cytokines, in particular tumor necrosis factor (TNF), may regulate muscle protein turnover during sep¬ 4
sis, their exact roles in this metabolic response remain to be defined. The influence of TNF on muscle protein breakdown is controversial. In several studies, TNF failed Accepted
for publication November 2, 1991. Department of Surgery (Drs Zamir, Hasselgren, Frederick, Higashiguchi, and Fischer), University of Cincinnati (Ohio) Medical Center, and the Department of Pathology (Dr Kunkel), University of Michigan Medical School, Ann Arbor. Presented at the 11th Annual Meeting of the Surgical Infection Society, Fort Lauderdale, Fla, April 9, 1991. Reprint requests to Department of Surgery, University of CincinFrom the
nati, 231 Bethesda Ave, Cincinnati, OH 45267-0558 (Dr Hasselgren).
protein
muscles5"8
or
MATERIALS AND METHODS Male
and
allowed to acclimatize in groups of three per cage in with a 12-hour dark-light cycle at 25°C for 3 days before
were
a room
experiments began. Two series of experiments were performed. In the first, the ef¬ fect of human recombinant TNF- (rTNF- ) on muscle protein turnover was examined. The cytokine preparation (Knoll Phar¬ maceuticals, Whippany, NJ) had a specific activity of greater than 5 x 106 U/mg of protein and an endotoxin content of less than 5 endotoxin units/mg of protein. A total amount of 300 µg/kg of body weight of the cytokine was administered intraperitoneally
in three divided doses at 4 PM, midnight, and 8 AM. Control rats received corresponding volumes (1 mL/100 g of body weight) of solvent (phosphate-buffered saline). Animals were fasted but had unlimited access to water after the first TNF or control in¬ jection in order to eliminate the effect of anorexia, induced by TNF, on muscle protein metabolism. In control experiments, rTNF- , heat inactivated at 100°C for 60 minutes was used in an identical protocol. Protein synthesis and degradation rates were measured in incubated extensor digitorum longus muscles (see below) 2 hours after the last TNF or control injection. In the second series of experiments, sepsis was induced by cecal ligation and puncture (CLP).2-16 Nonseptic rats underwent
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 06/10/2015
that is, laparotomy and handling, but no liga¬ tion or puncture of the cecum. All animals were resuscitated with
sham-operation,
normal saline (5 mL/100 g of body weight injected subcutaneously on the back at the time of CLP or sham-operation. The rats were fasted but had unlimited access to water after the operative procedures. Metabolic studies were performed in incubated mus¬ cles (see below) 16 hours after CLP or sham-operation. This time point was chosen on the basis of previous studies in which we found that muscle protein breakdown was increased and protein synthesis was inhibited 16 hours after CLP in rats.1617 One group of rats received an intraperitoneal injection of TNF antiserum (1 mL/100 g of body weight) 2 hours before CLP or shamoperation. Another group of rats received corresponding vol¬ umes of control serum 2 hours before CLP or sham-operation. The antiserum was prepared from rabbits injected with mouse rTNF- .18 Control serum was obtained from nonimmunized rab¬ bits. The serum samples were heated to 56°C for 30 minutes to inactivate the complement molecules and were then stored at -70°C until used. The antiserum had no cross-reactivity with murine interleukin-1, IL-lß, IL-6, or lymphotoxin.18 The ability of the TNF antiserum to cross-react with rat TNF and its effective¬ ness to neutralize rat TNF were reported previously.19 For the study of protein turnover rates, the extensor digitorum longus muscles with the tendons intact were gently dissected. The dissection was performed under light ether anesthesia. The muscles were tied by their tendons on stainless steel supports at approximate resting length, weighed, and incubated for 2 hours.20 Protein synthesis rate was determined by measuring in¬ corporation of tritiated phenylalanine into tricnloroacetic acid precipated proteins.17 Total and myofibrillar protein breakdown rates were determined by measuring the release of tyrosine and 3-methylhistidine, respectively, taking changes in tissue levels of the amino acids during incubation into account.20 Tyrosine and
3-methylhistidine
were
chromatography.2-20
assayed by high-performance liquid
Results are presented as means ±SEMs. For statistical com¬ parisons, Student's f test for unpaired observations was used in the first series of experiments and analysis of variance followed by Duncan's test was used in the second series of experiments. Survival rates were compared with 2 test.
RESULTS No animals died in the first series of experiments, but mild symptoms of illness in the form of lethargy, loose stools, and piloerection developed in the rTNF-a-treated rats. At autopsy, performed after muscles had been har¬ vested, that is, 2 hours after the last cytokine injection, there was no gross evidence of gut ischemia or hemor¬
rhage.
Treatment with rTNF- did not
significantly affect the
protein synthesis rate in incubated extensor digitorum longus muscles although there was a trend toward reduced protein synthesis rate following administration of the cytokine (Fig 1). Total protein breakdown rate was increased by 28% and the myofibrillar protein degrada¬
tion rate was increased threefold following administration (Fig 1). Administration of heat inactivated rTNF- did not affect muscle protein synthesis or degra¬ dation rates (data not shown), suggesting that the effect of the cytokine preparation was not caused by the small amount of endotoxin present in the preparation. In the second series of experiments, 25% of septic rats pretreated with control rabbit serum (n 32) died within 16 hours after CLP. The corresponding figure for septic rats that were pretreated with TNF antiserum ( 40) was 5% (P
role of tumor necrosis factor (TNF) in the regulation of muscle protein turnover was studied in rats. Protein synthesis and total and myofibrillar protein breakdown rates were measured in incubated extensor digitorum longus muscles. Intraperitoneal administration of recombinant TNF-\g=a\(300 \g=m\g/kgof body weight) increased total and myofibrillar protein breakdown rates by 28% and threefold, respectively, with no effect on protein synthesis. In subsequent experiments, sepsis was induced by cecal ligation and puncture or a sham-operation was performed. Rats received TNF antiserum (1 mL/100 g of body weight) or control serum 2 hours before cecal ligation and puncture or shamoperation. Treatment with TNF antiserum reduced the mortality rate from 25% to 5% following cecal ligation and puncture. The treatment had no effect on protein synthesis but reduced total and myofibrillar protein breakdown rates by 26% and 39%, respectively, in septic animals. Results suggest TNF is involved in the regulation of sepsis-induced muscle proteolysis.
metabolism when added to incubated when administered in vivo.9 In contrast, other reports suggest that TNF can induce muscle catabolism,10"12 possibly through stimulation of or interac¬ tion with adrenocortical hormones.1314 Furthermore, even if TNF can stimulate muscle protein breakdown when administered to nonseptic subjects, it does not necessar¬ ily mean that TNF actually participates in the regulation of muscle proteolysis during sepsis. The purpose of the present study was twofold. First, we tested the hypothesis that administration of TNF to nor¬ mal rats results in a sepsislike metabolic response in skel¬ etal muscle, that is an increased breakdown of predomi¬ nantly myofibrillar proteins. Second, we tested the hypothesis that TNF plays a role in the regulation of muscle proteolysis during sepsis by measuring muscle protein turnover rates in septic rats that had been treated with antiserum against TNF.
catabolism is a characteristic metabolic re¬ sponse to sepsis that mainly reflects increased pro¬ tein breakdown, especially myofibrillar protein break¬ down.1-2 Although this response may be beneficial to the organism during the early phase of sepsis by providing the liver with increased amounts of amino acids for
Sprague-Dawley rats (Zivic-Miller, Zelienople, Pa) weighing from 40 to 60 g were used. Rats of this size possess ex¬ tensor digitorum longus muscles that are thin enough to allow adequate diffusion of oxygen and substrates into tissue during incubation.15 The animals were maintained on regular rat chow,
\s=b\ The
to affect
(Arch Surg. 1992;127:170-174)
Muscle
acute-phase protein synthesis,3 a protracted degradation of muscle proteins will result in muscle wasting, fatigue, and delayed recovery. Recent studies suggest that both glucocorticoids and different cytokines, in particular tumor necrosis factor (TNF), may regulate muscle protein turnover during sep¬ 4
sis, their exact roles in this metabolic response remain to be defined. The influence of TNF on muscle protein breakdown is controversial. In several studies, TNF failed Accepted
for publication November 2, 1991. Department of Surgery (Drs Zamir, Hasselgren, Frederick, Higashiguchi, and Fischer), University of Cincinnati (Ohio) Medical Center, and the Department of Pathology (Dr Kunkel), University of Michigan Medical School, Ann Arbor. Presented at the 11th Annual Meeting of the Surgical Infection Society, Fort Lauderdale, Fla, April 9, 1991. Reprint requests to Department of Surgery, University of CincinFrom the
nati, 231 Bethesda Ave, Cincinnati, OH 45267-0558 (Dr Hasselgren).
protein
muscles5"8
or
MATERIALS AND METHODS Male
and
allowed to acclimatize in groups of three per cage in with a 12-hour dark-light cycle at 25°C for 3 days before
were
a room
experiments began. Two series of experiments were performed. In the first, the ef¬ fect of human recombinant TNF- (rTNF- ) on muscle protein turnover was examined. The cytokine preparation (Knoll Phar¬ maceuticals, Whippany, NJ) had a specific activity of greater than 5 x 106 U/mg of protein and an endotoxin content of less than 5 endotoxin units/mg of protein. A total amount of 300 µg/kg of body weight of the cytokine was administered intraperitoneally
in three divided doses at 4 PM, midnight, and 8 AM. Control rats received corresponding volumes (1 mL/100 g of body weight) of solvent (phosphate-buffered saline). Animals were fasted but had unlimited access to water after the first TNF or control in¬ jection in order to eliminate the effect of anorexia, induced by TNF, on muscle protein metabolism. In control experiments, rTNF- , heat inactivated at 100°C for 60 minutes was used in an identical protocol. Protein synthesis and degradation rates were measured in incubated extensor digitorum longus muscles (see below) 2 hours after the last TNF or control injection. In the second series of experiments, sepsis was induced by cecal ligation and puncture (CLP).2-16 Nonseptic rats underwent
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 06/10/2015
that is, laparotomy and handling, but no liga¬ tion or puncture of the cecum. All animals were resuscitated with
sham-operation,
normal saline (5 mL/100 g of body weight injected subcutaneously on the back at the time of CLP or sham-operation. The rats were fasted but had unlimited access to water after the operative procedures. Metabolic studies were performed in incubated mus¬ cles (see below) 16 hours after CLP or sham-operation. This time point was chosen on the basis of previous studies in which we found that muscle protein breakdown was increased and protein synthesis was inhibited 16 hours after CLP in rats.1617 One group of rats received an intraperitoneal injection of TNF antiserum (1 mL/100 g of body weight) 2 hours before CLP or shamoperation. Another group of rats received corresponding vol¬ umes of control serum 2 hours before CLP or sham-operation. The antiserum was prepared from rabbits injected with mouse rTNF- .18 Control serum was obtained from nonimmunized rab¬ bits. The serum samples were heated to 56°C for 30 minutes to inactivate the complement molecules and were then stored at -70°C until used. The antiserum had no cross-reactivity with murine interleukin-1, IL-lß, IL-6, or lymphotoxin.18 The ability of the TNF antiserum to cross-react with rat TNF and its effective¬ ness to neutralize rat TNF were reported previously.19 For the study of protein turnover rates, the extensor digitorum longus muscles with the tendons intact were gently dissected. The dissection was performed under light ether anesthesia. The muscles were tied by their tendons on stainless steel supports at approximate resting length, weighed, and incubated for 2 hours.20 Protein synthesis rate was determined by measuring in¬ corporation of tritiated phenylalanine into tricnloroacetic acid precipated proteins.17 Total and myofibrillar protein breakdown rates were determined by measuring the release of tyrosine and 3-methylhistidine, respectively, taking changes in tissue levels of the amino acids during incubation into account.20 Tyrosine and
3-methylhistidine
were
chromatography.2-20
assayed by high-performance liquid
Results are presented as means ±SEMs. For statistical com¬ parisons, Student's f test for unpaired observations was used in the first series of experiments and analysis of variance followed by Duncan's test was used in the second series of experiments. Survival rates were compared with 2 test.
RESULTS No animals died in the first series of experiments, but mild symptoms of illness in the form of lethargy, loose stools, and piloerection developed in the rTNF-a-treated rats. At autopsy, performed after muscles had been har¬ vested, that is, 2 hours after the last cytokine injection, there was no gross evidence of gut ischemia or hemor¬
rhage.
Treatment with rTNF- did not
significantly affect the
protein synthesis rate in incubated extensor digitorum longus muscles although there was a trend toward reduced protein synthesis rate following administration of the cytokine (Fig 1). Total protein breakdown rate was increased by 28% and the myofibrillar protein degrada¬
tion rate was increased threefold following administration (Fig 1). Administration of heat inactivated rTNF- did not affect muscle protein synthesis or degra¬ dation rates (data not shown), suggesting that the effect of the cytokine preparation was not caused by the small amount of endotoxin present in the preparation. In the second series of experiments, 25% of septic rats pretreated with control rabbit serum (n 32) died within 16 hours after CLP. The corresponding figure for septic rats that were pretreated with TNF antiserum ( 40) was 5% (P
