Effect of insulin and leucine on protein turnover in rat soleus muscle after burn injury

E f f e c t o f I n s u l i n and L e u c i n e on P r o t e i n T u r n o v e r in Rat S o l e u s M u s c l e A f t e r Burn Injury Richard Odessey and Brian Parr To investigate the effects of thermal injury on muscle protein turnover, net protein breakdown and incorporation of leucine into protein was measured in vitro in rat soleus at 3 days following a 3 sec burn to one hindlimb. The weight gain and food consumption of the burn injured animals was similar to that of unburned animals. However, the burn caused an 1 1 % decrease in soleus muscle weight and protein content. The levels of ATP, phosphocreatine, and the phosphocreatine/ creatine ratio w e r e all normal. Net protein b r e a k d o w n from the burn-injured muscle was elevated 3 5 % while the incorporation of leucine into protein was unchanged. Thus the increase in protein breakdown in the muscle from the burned region appears to be responsible for the loss in muscle protein. Leucine oxidation was also stimulated by burn. Since protein turnover and leucine oxidation in the contralateral muscle of the burned animal was identical to those from unburned animals, the effects appear to result from direct thermal injury to the muscle from the burned hindlimb rather than from systemic alterations in the metabolic or endocrine environment. In addition, a physiological concentration of insulin ( 1 0 0 / ~ U / m l ) was found to stimulate incorporation of leucine into protein and inhibit net proteolysis to the same extent in soleus from burned end unburned limbs. While lower insulin concentrations need to be tested, there appears to be no evidence for insulin resistance with respect to protein turnover under these conditions.

' E G A T I V E nitrogen balance due to protein catabolism is characteristic of many types of severe trumatic injuries 13 and represents a serious clinical problem. Most of the urinary nitrogen under these conditions is ultimately derived from the accelerated breakdown of skeletal muscle protein. 4"5 Various therapeutic regimes have been used to arrest muscle wasting in trauma. Although the anabolic effects of insulin are well known, its use in trauma victims has not been entirely successful. 6-8 Branched chain amino acids and leucine in particular are unique in the ability to inhibit protein breakdown and stimulate protein synthesis in skeletal and heart muscle under certain conditions. 9-13 Some animal and human data suggest that branched chain amino acid infusion can in fact markedly reduce the negative nitrogen balance observed in victims of severe trauma and infections. 6'8'15-17 However, in vivo many factors including caloric balance, circulating hormones, environmental temperature contribute to the injury response. 1'~9Little is known of the direct effects of the branched chain amino acids on skeletal muscle from trauma victims. In addition, several investigators 2°'21 have observed that in skeletal muscle, some of the metabolic effects in burn trauma may arise from direct thermal injury as well as alterations in the

N

From the University o f Virginia Medical Center, Charlottesville, Va. Received for publication March 17, 1981. Supported in part by BMRSG 5 S 0 7 RR05431-18 and an N S F fellowship (SPI78-21278) to Brian Parr. Presented in part at a meeting o f the American Physiological Society, Toronto, Canada, October, 1980. Address reprint requests to Dr. Richard Odessey, Department o f Physiology, LSU Medical Center, 1910 Perdido Street, New Orleans, La. 70112. © 1982 by Grune & Stratton, Inc. 00 2 6 ~ 4 9 5/8 2/3101~9014 501.00/0 82

endocrine environment. The experiments to be presented were undertaken to examine the direct effects of the branched chain amino acids and insulin on the regulation of protein turnover in burn-injured skeletal muscle under controlled nutritional conditions. MATERIALS A N D METHODS Male Sprague Dawley rats (45-50 g) were obtained from Hilltop Lab Animals (Scottdale, Pa.). In the course of a single experiment, animals from the same litter were used. Chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.) and Fisher Scientific Co. (Pittsburgh, Pa.). [1-~4C] Leucine was purchased from New England Nuclear Corp. (Boston, Mass.). Using the protocol described by Turinsky and Shangraw 2°'2~ animals were divided into two groups. One group was anaesthetized with ether and one hindlimb was scalded by immersion in a 90°C water bath for 3 sec. This procedure has been reported to produce a subcutaneous temperature of 53°C and a deep muscle temperature of 49°C at 15 sec postburn. 21 The second group (unburned) was anaesthesized but not scalded. All animals were subsequently housed in individual cages and fed equal amounts of Purina Laboratory Chow. The scalding procedure produces an anaesthetizing full thickness burn, and therefore no further anaesthesia was administered during the recovery period. It should also be noted that human patients suffering from this type of burn also do not require any analgesic medication during recovery. ~4 Soleus muscles were obtained from small rats (60-65 g at the time of dissection) because it is critically important to have thin muscles to allow adequate diffusion of substrates and oxygen. Rats were killed by stunning and cervical dislocation. The muscles were rapidly removed according to the procedure of Maizels et al. 22 and tied to stainless steel wire supports and maintained at approximately rest length. To prevent the alteration in ion gradients which may occur upon dissection, TM the muscles were temporarily placed in ice-cold buffer (HEPES (10 mM, pH 7.8), KCI 120 mM, NaC1 20 mM, MgCI: 0.5 mM and KH2PO4 0.5 mM) and then transferred to an Erlenmeyer flask containing Krebs Ringer bicarbonate buffer with 5 mM glucose and 0.13% bovine serum albumin equilibrated with O2/CO2 (95:5). Where noted, [1-~4C] leucine and/or insulin was present in the medium. After 45 min of preincubation at 37°C the solei were transferred to new flasks with medium of the same composition. After a 1 hr incubation the muscles were removed, Metabolism, Vol. 31, No. 1 (January), 1982

84

ODESSEY A N D PARR

Table 3. Effect of Leucine on Protein Breakdown by Rat Soleus

Table 4. Effect of Burn Injury on [1-1~C] Leucine Metabolism by

Muscle Subject t o B u r n I n j u r y

Rat Soleus

Net Twosine Release Leucine Conc (mM):

0.0

0.5

2.5

~4c

~4C0 2

in Protein

Production

nmole/mg w e t w t / h r

nmole/mg wet wt/hr Burned

.327 ± . 0 3 3 "

. 3 3 7 _+ . 0 6 5 t

.264 ± .028"

Contralateral

.243 ± .020

. 1 5 6 _+ .024:[:

. 1 6 5 ± .011

Unburned

.241 _+ . 0 1 7

.161 + .012:1:

. 1 7 7 _+ .015:1:

Burned

0 . 3 5 8 -+ .026

1.94 _+ . 1 9 "

Contralateral

0.409 ± .024

1.38 + . 11

Unburned

0 . 3 6 4 _+ . 0 1 8

1.30 _+ . 10

* p < .01 versus contralateral,

* p < .05 versus contralateral.

Muscles were preincubated as described in Table 1 in the presence of

t P < .01 versus contralateral.

2.5 m M leucine. After 4 5 min they were transfered to fresh medium

~p < .01 versus no leucine,

containing [1J4CI leucine (2.5 m M , 0 . 0 7 #Ci/umole), Then after 1 hr

Soleus musc)es were incubated as described in Table 1 with 0, 0 . 5

incubation the muscles were removed and homogenized in PCA. The

m M or 2 . 5 m M leucine added to the incubation medium. Thereafter the

precipitated protein was washed and counted. The flask was restoppered

medium was assayed for tyrosine. All values represent the mean +

and injected with acid to collect 14CO2. Each value represents the

s.e,m, of 7 - 1 3 muscles.

mean _+ s.e.m, of B muscles.

To study protein turnover, the release of tyrosine into the incubation medium was measured. Previous studies have shown that this amino acid is neither degraded or synthesized by skeletal muscle and its intracellular pool remains constant during the incubation period. 27 Therefore release of tyrosine is a reflection of net protein breakdown occurring in the muscle. As shown in Table 3, net protein breakdown was significantly elevated (35%) in the burn injured soleus which is consistent with the observed loss of muscle protein. Note that in the contralateral (unburned) leg tyrosine release is identical to that in unburned animals. Since leucine is known to inhibit protein turnover in skeletal muscle 9-~2 the effect of this amino acid was tested. As expected, 0.5 m M leucine markedly inhibited (35%) tyrosine release from the contralateral and unburned muscles and 2.5 mM levels had no further effect (Table 3). However, 0.5 m M leucine had no significant effect on net protein breakdown in the burn injured muscle. Even at 2.5 mM no significant difference was observed. Net protein breakdown was still 60% greater in burned vs. the contralateral limb. The incorporation o f [1-14C] leucine into protein was also measured. A high concentration of leucine

(2.5 mM) was used to eliminate any effects due to changes in intracellular specific activity. 26 Under these conditions amino acid incorporation into protein (Table 4) was not significantly changed by burn injury. Using 0.5 mM leucine similar results were obtained (Table 5). Previous studies have reported that increases in skeletal muscle protein catabolism are often accompanied by increases in branched chain amino acid catabolism. 28'29 The data in Table 4 show a clear and significant increase of 40% in leucine catabolism by burn-injured tissue. As with tyrosine release, this appears to be a local effect of the burn injury. To study the effects of insulin on bUrn-injured soleus 100/~U/ml of the hormone (within the physiological range for the rat) was added to the incubation medium. As shown in Table 5, insulin significantly stimulated the incorporation of leucine into protein and significantly decreased net protein breakdown in injured and normal muscles. The percent changes in protein metabolism caused by insulin were similar in burned and unburned tissue. Thus even in the presence of insulin differences in net protein catabolism remain. At this concentration of insulin there is no evidence of insulin "resistance."

Table 5. Effect of Insulin on Protein Turnover in the Presence of Leucine (0.5 m M ) Net Protein Breakdown (Tyr Release) Insulin (IO0/LU)

-

Amino Acid Incorporation (14C in Protein) +

-

-F

nmole/mg/wet wt/hr Burned

. 3 3 7 _+ . 0 6 5

. 2 5 5 _+ . 0 1 2

, 1 7 3 _+ . 0 0 9

.217 ± . 0 1 2 *

Contralateral

.156 + .024

(--24%) .115 ± .012"

.186 ± . 0 0 9

(+25%) . 2 3 3 _+ . 0 0 7 "

Unburned

.161 _+ . 0 1 2

(--26%) . 1 1 4 _+ . 0 1 2 "

. 1 9 5 _+ .007

(+25%) ,217 ± .007*

(--29%)

( + 11%)

* p < .05 versus No insulin. Muscles were incubated as described in Table 4. Insulin was present at 1 0 0 / L U / m l where noted. Tyrosine release and incorporation of 14C-leucine into protein were measured as described. Each value represents the mean _+ s.e.m, of 6 muscles.

PROTEIN TURNOVER IN BURNED MUSCLE

blotted, weighed and homogenized in 1 ml of ice-cold 3N perchloric acid (PCA) with 0.5 m M EDTA. When tissue metabolites were assayed, the muscles were immediately frozen in Wollenberger tongs precooled in liquid nitrogen. The medium was deproteinized with PCA and both tissue and medium samples were centrifuged. Supernatants were neutralized with 3N KOH, 0.25 M MES and 0.25 M MOPS. After removal of the perchlorate precipitates solutions were stored at - 8 0 ° C until analysis. PCA precipitates were washed with ethanol-ether (1:1 v/v) and dissolved in 1N NaOH. Protein was determined by the Biuret procedure 23 with bovine serum albumin as a standard. Tyrosine, 24 ATP, creatine phosphate, creatine, pyruvate, and lactate were assayed fluorometricallyY ~4CO2 production and t4C incorporation into protein was measured as described previously.26 Calculations were made using the specific activity of leucine in the medium. Student's t test was used in statistical analyses of the data. Burned and unburned contralateral muscles from the same animal were used in paired analysis. Values are reported as the mean ± standard error of the mean (S.E.M.).

RESULTS

The burn injury resulted in 0% mortality after 3 days in over 75 animals. Burned and unburned rats had similar weight gains (approx. 16 g/3 days) and food intake was also similar. The skin tissue of the burned leg showed varying degrees of dermal inflammation, edema and scar tissue. The general appearance of the muscle from the burned leg was similar to that of the control. However the protein content of the soleus muscle in the burned limb declined 11% after 3 days (Fig. 1). Since the protein/wet weight ratio (0.17 _+ .01) was identical in muscles from both limbs, these findings indicate a significant loss of muscle

O CONTRALATERAL 70

| "I= o .Q

m

BURNED

IlUNUNtD 60

"1-

.,g

~p o,

50

83

Table 1. High Energy Phosphate Levels in Normal and B u r n I n j u r e d S k e l e t a l Muscle

Treatment

Phosphoereatine

ATP

Creatine

PCr/Cr

nmole/mg w e t weight Burned

3.7 _+ 0 . 2

10.2 _+ 1.1

13.6 _+ 0 . 9

Contralateral

3 . 4 _+ 0 . 2

9 . 4 _+ 0.7

12.5 _+ 1.0

0 . 7 5 _+ , 0 4 0 . 7 5 _+ .11

Unburned

3.7 _+ 0 . 2

9.8 _+ 0 . 5

12.1 _+ 0 . 9

0.81 ± .06

Rat soleus muscles were mounted on wire supports at rest length and preincubated at 3 7 ° C in Krebs-Ringer bicarbonate buffer containing 5 m M glucose and 0 . 1 3 %

albumin. After 4 5 min the muscles were

transfered to flesh medium and incubated 1 hr. A t the end of the incubation muscles were frozen using Wollenberger tongs cooled in liquid nitrogen and processed for assay of metabolites. There were no significant differences between burned and unburned muscles. Each value represents the mean _+ s.e.m, of 8 - 1 0 muscles.

protein from the burn and little or no evidence for muscle edema. Although the burn-injured muscle showed signs of atrophy, ATP levels were similar to that of control (Table 1). In addition, creatine phosphate, creatine and the creatine phosphate/creatine ratio which are sensitive indicators of tissue energy status were also normal in the burn-injured tissues (Table 1). These values are in close agreement with those obtained when the soleus was removed and frozen within 30 sec of death (ATP = 3.75 _+ .14 t~mole/g, creatine phosphate = 10.6 _+ 0.6 umol/g) and were also similar to values reported by others for the soleus muscleY Therefore oxygenation and energy metabolism appear adequate. In addition, the preparation gave linear rates of all metabolic parameters measured (see below). As has been reported previously, glycolysis was somewhat elevated in burn injured soleus muscle compared to the contralateral muscle (Table 2). The stimulation of lactate and pyruvate release (about 45%) was much less than that observed by previous investigators. 2° Furthermore, compared to muscles from unburned animals the stimulation of glycolysis was not significantly different. Note also that the lac/pyr ratio of 10:11 was low indicating a high oxidation state characteristic of well oxygenated tissue. Table 2. Effect o f B u r n i n j u r y On Glycolysis in Rat Soleus

o E 40

Treatment

Pyruvate Release

Lactate Release

LAC/PYR Ratio

nmole/mg w e t w t / h r

3¢ Fig. 1. Soleus muscle size 3 days postburn. The protein content of the burned muscle is significantly less ( p < .001 ) than both the contralateral muscle and the muscles from the unburned animals. A t least 14 muscles w e r e measured for each g r o u p .

Burned

1.39 _+ .17

16.2 _+ 1 . 6 "

11.7

Contralateral

1.11 +_ .16

11.4 _+ 2.1

10.3

Unburned

1.42 + .OB

14.2 ± 1.3

10.0

* p < .05 versus contralateral. Soleus muscles were incubated as described in Table 1. Thereafter the medium was mixed with PCA and processed for assay of pyruvate and lactate. Each value represents the mean _+ s.e.m, of 7 - 1 4 muscles.

PROTEIN TURNOVER IN BURNED MUSCLE

DISCUSSION

These experiments demonstrate that mild thermal injury results in muscle atrophy accompanied by an increase in net breakdown of tissue protein. The tissue atrophy is most likely due to an increased rate of protein breakdown (Table 3) since in the presence of high levels of leucine (Table 4) there was no difference in protein synthesis between burned and unburned muscle tissue. These experiments do not exclude a burn-induced change in protein synthesis, since this was not tested in the absence of leucine. However any impairment of synthesis, if it exists, is corrected by the presence of leucine. These findings are consistent with observations that in human patients with major burns, whole body protein breakdown is elevated. Patients suffering from multiple skeletal trauma, sepsis, and major surgery also exhibit a marked increase in whole body protein degradation. 4'16'32'33'36 The changes in protein breakdown and leucine catabolism (Tables 3, 4) appear to be a local effect of thermal injury since the contralateral muscle from the same animal was unchanged from normal, Furthermore there is no differential effect of insulin on burned and contralateral muscle. These findings raise the intriguing possibility that systemic effects of burn on muscle are independent and may be different from the response of muscle tissue sustaining direct thermal injury. A similar distinction has also been proposed with respect to the effect of burn on muscle glucose metabolism. 2~ This hypothesis is consistent with the data of Frayn and Maycock 41 who found no change in protein catabolism of unburned leg muscles in vitro after a 20% scald to the mouse dorsum. This hypothesis may also explain the failure to observe a change in protein breakdown after minor surgery and skeletal trauma. 37-~° Where direct trauma to muscle tissue is small and systemic effects of uninjured tissue would be expected to predominate, a decline in protein synthesis (rather than an increase in proteolysis) is observed both in vitro and in v i v o . 37-41 While the hormonal milieu cannot account for the in vitro effects of the burn injury, decreased use of the injured leg may have contributed to the observed atropy. 3°'3~ This possibility is under investigation. However a recent report suggests that this may be a minor factor. 34 It has been well established (9-13, 41, Table 3) that leucine inhibits net protein breakdown in muscles from uninjured animals and in uninjured muscles from burned animals. This anabolic effect occurs in the physiological range (0.1 to 0.5 mM) 42 and changes of plasma leucine concentration of 20%-30% in muscle or 25%--50% in plasma result in a marked improvement in nitrogen balance in patients receiving amino

85

acid infusions. 43 Thus the failure of leucine to inhibit proteolysis only in muscles sustaining direct thermal injury has potential clinical implications. Perhaps in vivo the efficacy of amino acid therapy is mediated through effects on uninjured muscle. Further work is necessary to clarify the basis of branched chain amino acid therapy as a treatment for stress-induced protein catabolism. Several investigators ~3'42have suggested that a product of leucine degradation is the mediator of its antiproteolytic effect. It is therefore of interest that leucine oxidation is elevated in the burn-injured muscle (Table 4) as it is during other types of s t r e s s . 32'44'45 Perhaps the burn-induced increase in leucine catabolism is responsible for the inhibition of the antiproteolytic effect. The effects of leucine on protein breakdown were not observed by Turinsky and Shangraw 2°'47 who used a similar model of burn injury. However, these workers studied much older and larger rats (200 g). Li and Jefferson 12 have reported that age and nutritional status may affect the protein-sparing ability of the branched chain amino acids. Also muscles were incubated unstretched in buffer without protein. Under these conditions soleus muscle will swell and diffusion distances for these large muscles will be quite long. It is therefore likely that such muscles would have hypoxic cores and diffusion of substrates and removal of metabolites during the incubation might be slowed or impaired. These conditions may contribute to the large increase in glycolysis in burned muscles. The failure to observe an inhibition of protein breakdown by leucine may indicate a failure of this amino acid to penetrate the cells or be metabolized sufficiently to affect these processes. Since the nitrogen-sparing effect of the branched chain amino acids is well documented in in vivo studies of both animals and man, it appears that a rat model which exhibits this phenomenon should have more application to elucidating the underlying mechanisms of protein-sparing therapy. Burned and unburned muscles responded equally to the anabolic effects of insulin (Table 5). Insulin also stimulated protein synthesis in soleus muscle of mice receiving a 20% dorsal burn to the same extent as in muscle from control animals. 41 These findings would suggest that insulin resistance with respect to protein synthesis (if it exists) is not an inherent property of skeletal muscle from burn-injured animals. While experiments at lower insulin concentrations will be necessary to confirm this observation, the evidence supports the suggestion that the insensitivity to insulin in burns may be mediated by circulating factors. 35 In the rat soleus insulin stimulating of protein

86

ODESSEY AND PARR

synthesis alone may account for its effect on net protein breakdown (Table 5). In fact, much higher concentrations of insulin are necessary to slow proteolysis (500/~U/ml) than to stimulate protein synthesis (25-100 # U / m l ) . 4l'46 Thus in vivo, the action of insulin

may be primarily to stimulate protein synthesis. The branched chain amino acids may play an important physiological role in keeping muscle protein breakdown in check.

REFERENCES

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PROTEIN TURNOVER IN BURNED MUSCLE

protein synthesis and breakdown rates in children before and after reconstructive surgery of skin. Metabolism 27:27-34, 1978 39. Moldawer LL, O'Keefe SJD, Bothe A, et al: In vivo demonstration of nitrogen-sparing mechanisms for glucose and amino acids in the injured rat. Metabolism 29:173-180, 1980 40. O'Keefe SJD, Sender PM: "Catabolic" loss of body nitrogen in response to surgery. Lancet: 1035-1037, 1974 41. Frayn KN, Maycock PF: Regulation of protein metabolism by a physiological concentration of insulin in mouse soleus and extensor digitorum longus muscles. Biochem J 184:323-330, 1979 42. Tischler ME, Goldberg AL: Relationship of leucine catabolism to its regulatory effects on protein turnover in muscle. Fed Proc 39:1682, 1980 43. Freund H, Yoshimura N, Lunetta L, et al: The role of the

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branched-chain amino acids in decreasing muscle catabolism in vivo. Surgery 83:611-618, 1978 44. Ryan NT: Metabolic adaptations for energy production during trauma and sepsis. Surg Clin N Am 56:1073-1090, 1976 45. Ryan NT, George BC, Odessey R, et al: The effect of hemorrhagic shock, fasting and eorticosterone administration of leucine oxidation and incorporation into protein by skeletal muscle. Metabolism 23:901-904, 1974 46. Jefferson LS, Li JB, Rannels SR: Regulation by insulin of amino acid release and protein turnover in the perfused rat hemicorpus. J Biol Chem 252:1476-1483, 1977 47. Shangraw RE, Turinsky J: Effects of leucine on thermally injured skeletal muscle. Physiologist 23:94, 1980