Glutamine metabolism in skeletal muscle of septic rats

Glutamine

Metabolism

in Skeletal Muscle of Septic Rats

M. Salleh M. Ardawi and May F. Majzoub The metabolism of skeletal muscle glutamine was studied in rats made septic by cecal ligation and puncture technique. Blood glucose was not significantly different in septic rats, but lactate, pyruvate, glutamine, and alanine were markedly increased. Conversely, blood ketone body concentrations were markedly decreased in septic rats. Both plasma insulin and glucagon were markedly elevated in septic rats. Sepsis increased the rates of glutamine production in muscle, but without marked effects on skin and adipose tissue preparations, with muscle production accounting for over 87% of total glutamine produced by the hindlimb. Sepsis produced decreases in the concentrations of skeletal muscle glutamine, glutamate, 2-oxoglutarate, and adenosine monophosphate (AMP). The concentrations of ammonia, pyruvate, and inosine monophosphate (IMP) were increased. Hindlimb blood flow showed no marked change in response to sepsis, but was accompanied by an enhanced net release of glutamine and alanine. The maximal activity of glutamine synthetase was increased only in quadriceps muscles of septic rats, whereas that of glutaminase was decreased in all muscles studied. Tyrosine release from incubated muscle preparation was markedly increased in septic rats; however, its rate of incorporation was markedly decreased. lt is concluded that there is an enhanced rate of production of glutamine from skeletal muscle of septic rats. This may be due to changes in efflux and/or increased intracellular formation of glutamine; these suggestions are discussed. Copyright 0 1991 by W.B. Saunders Company

C such as bums

SEPSIS may develop after major trauma or abdominal surgery; the septic episode is not merely limited to the bacterial insult, but has been described as an acquired disease of intermediary metabolism.‘*2 The changes in metabolism include enhanced muscle proteolysis, increased nitrogen flw, increased nitrogen loss, increased rates of fatty acid mobilization, and increased or decreased rates of gluconeogenesis.3’9 Glutamine is the most abundant amino acid in the body. It has the highest concentration in the plasma, and it accounts for over 50% of the intracellular amino acid content.” Skeletal muscle has been shown to produce glutamine,“-” and unlike other amino acids produced or utilized by skeletal muscle, glutamine does not undergo reversible transamination; instead its net production by muscle is controlled by the balance of flux through glutamine synthesis and degradation.‘4.‘5 It is well established that the concentration of glutamine of skeletal muscle is decreased in various catabolic conditions, including injury, surgery, uncontrolled diabetes, sepsis, or burns.7,8.‘6,‘7This decrease occurs concomitantly with an increase in the net rate of protein degradation in skeletal muscle.” Several possible mechanisms have been suggested to explain the decrease in glutamine concentration in skeletal muscle. These include changes in amino acid transport across skeletal muscle plasma membrane,19 inhibition of intracellular glutamine formation,*” and changes in the rates of protein degradatior? and/or synthesis,23,24respectively. The present work was designed to obtain more information about the regulation of skeletal muscle glutamine metabolism in septic rats with cecal ligation and puncture technique. Hence, to provide further information about the metabolic response of glutamine to sepsis, the extent of its release by skeletal muscle, skin, and adipose tissue were measured. The key metabolites in the glutamine biosynthetic pathway, together with plasma amino acids, were determined. Moreover, the effect of sepsis on skeletal muscle glutamine concentration, efflux, and synthetic and degradation enzyme activities, as well as protein synthesis and degradation, also were investigated.

MATERIALS AND METHODS

LINICAL

Metabolism,

Vol40, No 2 (February), 1991:

pp 155-164

Animals

This study was conducted in accordance with the National Institutes of Health guidelines for the use of experimental animals. Male Wistar albino rats (190 to 220 g) were supplied by The Animal House of King Fahd Medical Research Centre, College of Medicine and Allied Sciences, King Abdulaziz University, Jeddah, Saudi Arabia. Rats were maintained on a standard diet (commercial rat cubes containing [wt/wt] approximately 18% protein, 3% fat, 77% carbohydrate, and 2% of an inorganic-salt mixture with a vitamin supplement [Grain Silos and Flour Mills Organization, Jeddah, Saudi Arabia]) and water ad libitum. Animals were kept in a controlled environment (constant temperature 24”C, and a 12-hour light-dark cycle). Animals were starved for 48 hours after induction of sepsis (see below) or laparotomy (sham-operated), but allowed free access to water and housed individually. Body weight, volume of urine, and weight of feces were recorded daily. Animals were killed by cervical dislocation 48 hours after cecal ligation or sham-operation. In the present work, sepsis was induced via cecal ligation and puncture technique.” All operations were performed under pentobarbital (40 mgikg body weight) anesthesia. A midline laparotomy was performed in the sham-operated animals; the cecum was mobilized by incising the mesocecum, and was then returned to the abdominal cavity. In the septic groups, after mobilization of the cecum, the feces were milked into the cecum, which was then ligated with a single 3.0-silk ligature in such a manner that the bowel continuity was maintained. The antimesenteric surface of the cecum was punctured once with a 21-gauge needle, and the bowel was returned to the abdominal cavity. The abdominal wall was closed in two layers and all rats received 0.9% (wt/vol) NaCl (2.5 mUlO body weight) subcutaneously. With this procedure, an

From the Department of Clinical Biochemistv and the Clinical Metabolic Research Laboratory, King Fahd Medical Research Centre, College of Medicine and Allied Sciences, King Abdulaziz University Jeddah, SaudiArabia. Supported by Universi~ grant (No. 026l408) to M.Xh4.A. at the Clinical Metabolic Research Laboratory. Address reprint requests to M. Salleh M. Ardawi, MD, PO Box 9029, feddah 21413, Saudi Arabia. Copyright 0 1991 by W.B. Saunders Company 0026-0495f91l4002-0008$03.00/0 155

156

experimental peritonitis is created in which the rats go through the hypermetabolic hyperdynamic septic state as described by Wichterman et al.zsA single puncture with a smaller needle gauge than that used previously was chosen so that the peritonitis could develop at a slower rate and allow animals to starve and survive for 48 hours.9.26Moreover, it was decided to use starved animals, since dietary nutrients cannot be absorbed in this model, and this is likely also to be the case in patients with intra-abdominal sepsis.” Chemicals and Enzymes All chemicals and enzymes were obtained from the same sources described previously.’ Rectal Temperature and Hemodynamic Measurements Rectal temperature of rats was measured using a temperature monitor (model Ellab, type te-3-5, Ellab, Copenhagen, Denmark) with thermoelectrodes. Three different temperature readings were made over 30 minutes for each rat and the mean value was taken. For the measurements of hemodynamic parameters, rats were anesthetized with ether, and a 22-gauge polytetrafluorethylene catheter was inserted into the left common carotid artery. Blood pressure and heart rate were recorded with a Gould P23ID transducer (Gould Recording Systems, Cleveland, OH). The mean arterial pressure was derived electronically through an integrator circuit. Cardiac output was measured by the thermodilution technique as described previously.*’ Muscle, Skin, and Adipose Tissue Incubations Animals were killed by cervical dislocation. Intact epitrochlearis muscles were rapidly removed, rinsed, blotted, and incubated in silicone-treated 25mL Erlenmeyer flasks containing 2.0 mL of Krebs-Henseleit bicarbonate buffeti” containing 1.2 mmol/L CaCl,, 5 mmol/L glucose, and 5 mmol/L 4-(2_hydroxyethyl)-piperazineethanesulphonic acid (pH 7.4). The gas phase was O&OZ (19:1, vol/vol) and the temperature was 37°C. Flasks were gassed for 5 minutes, stoppered, and incubated in a shaking water bath (Grant Instrument [Cambridge], Barington, Cambridge, England) at 100 to 110 oscillations per minute. After preincubation for 30 minutes, muscles were transferred to flasks with 2.0 mL of fresh incubation medium and again gassed for 5 minutes. Muscles were removed from the medium after 60 minutes of incubation, rinsed, blotted, and frozen in liquid nitrogen. Muscles and incubation media were stored at -130°C. Frozen muscle preparations and media were extracted as described previously.‘9 Shaved hindlimb skin slices (20 to 40 mg) were prepared by cutting pressed skin between two glass slides with a razor blade. Skin slices were incubated and extracted as described for epitochlearis incubations. Epididymal adipose tissue pads (80 to 120 mg) were incubated and extracted as described for epitochlearis incubations, except 1.0 mmol/L L-leutine was included in the incubation medium. The above preparations remained viable in vitro, as judged by adenosine triphosphate (ATP) and adenosine monophosphate (AMP) concentrations that were not different from concentrations in vivo. Plasma and Hindlimb Muscle Sampling Rats were anesthetized with ether, and blood was withdrawn from the abdominal aorta. For the determination of plasma amino acids, blood was centrifuged to remove erythrocytes, and the plasma was treated with ice-cold 4% (wtivol) sulphosalicylic acid: the precipitated proteins were removed by centrifugation, and the supernatant was adjusted to pH 2.2 with LiOH. Amino acids were determined with an LKB Amino Acid Auto-Analyzer (model 4151 Alpha Plus LKB-Produkter, AB-S-16126, Bromma, Sweden).

ARDAWI AND MAJZOUB

For the isolation of skeletal muscles of the hindlimbs. rats were anesthetized with pentobarbital (40 mgkg body weight) and the hindlimb muscles (biceps femoris, gastrocnemius, and underlying muscles) were exposed and freeze-clamped in liquid nitrogen. Frozen samples were weighed and ground into a powder in liquid nitrogen using a mortar and pestle. Samples were then homogenized with 5 vol of 4% (wt/vol) HCIO, using a Polytron homogenizer PCU-2 (Kinnematica GmbH, Kriens-Lucerne, Switzerland) at position 6 for 20 seconds at 0°C then centrifuged at 13,800 x g for 5 minutes as described previously.” The KC10 .,-free supernatant was used for metabolite determinations. Muscle extracts for amino acid analysis were prepared by homogenizing the frozen powder in 4% (wt/vol) ice-cold sulphosalicyclic acid as described above. Muscle Water and Chloride Determinations Total tissue water of incubated epitrochlearis muscles was determined in separate experiments. Muscles were dried at 115°C and the weight of dry fat-free tissues was determined after extraction in petroleum ether. Intracellular and extracellular waters were calculated by the method of Bergstrom.3’ Muscle and plasma chloride concentrations were measured with Jenway Digital chloride-meter (model PCLM3). In epitrochlearis muscles of sham-operated control rats, total tissue water, and intracellular and extracellular volumes (expressed as mL/kg muscle weight) were (means 2 SD): 765 2 11. 570 2 10, and 195 2 8, respectively. Muscle water content of septic rats was 759 2 13. Intracellular and extracellular volumes were 572 2 12 and 187 ? 10, respectively. Arteriovenous Concentration Difference and Hindlimb Blood Flow Measurements Blood flow measurements were performed as described by Ardawi.2’ Glutamine plus alanine exchange rates across the hindlimbs were calculated by multiplying hindlimb blood flow by the respective amino acid arteriovenous concentration difference. Preparation of Homogenate and Assay of Glutamine Synthetase and Glutaminase Animals were killed by cervical dislocation and various muscles (soleus, gastrocnemius, and quadriceps) were rapidly dissected and used for the measurements of the maximal activities of glutamine synthetase and glutaminase. Muscles were weighed and homogenized (using a Polytron homogenizer at position 6 for 15 to 20 seconds at 0°C) in 5 vol of extraction medium” and the activities of glutamine synthetase and glutaminase were measured as described previously.“,” Determination of Metabolites, Plasma Insulin and Glucagon Metabolites in neutralized extracts of muscles, media. and plasma were determined spectrophotometrically with a Beckman DU-6 recording spectrophotometer (Beckman Instruments, Palo Alto, CA) by standard enzymatic methods as described previously.X5 Plasma insulin and glucagon were measured using radioimmunoassay (RIA) technique and RIA kits were obtained from Diagnostic Products, Los Angeles, CA (for insulin) and from ICN Biomedicals, Carson, CA (for glucagon), respectively. Radioactivity was determined in a Beckman Gamma Counter, model 5500 (Beckman Instruments).

MUSCLE GLUTAMINE

METABOLISM

IN SEPSIS

157

Nitrogen Balance Measurements

Presentation of Results and StatisticalAnalysis

For the determination of the nitrogen balance of sham-operated and septic rats, animals were placed in metabolic cages that allowed the separate collection of urine and faeces after shamoperation or cecal ligation. Urine was collected during 24-hour periods (from 8:00 AMto 800 AM) in a vessel containing 0.5 mL of concentrated H,SO,. The 24-hour urine volume was measured and a sample was frozen at -130°C. Feces were collected at 24-hour intervals and weighed. The nitrogen content of urine, feces, and food was determined by the micro-Kjeldahl method.j6 Daily nitrogen input, excretion, and balance were determined during the experimental period (ie, 48 hours) for sham-operated and septic rats.

Data are presented as means 2 SD for the number of animals indicated in each table. Glutamine and alanine production rates by in vitro tissue preparations were calculated by the following equation: Production rate = [glutamine release to the medium] + [tissue concentration following incubation - mean tissue concentration after preincubation]. Blood flow values were expressed as mWmin/lOO g body weight and substrate or metabolite exchange rates by the hindlimb were expressed as nmol/min/lOO g body weight. Glutamine synthetase and glutaminase activities were expressed as nmol of glutamine and glutamate formed/mitt/g wet weight, respectively, or as nmol/min/mg of protein. Where appropriate, comparisons between sets of data were made using Student’s t test.

Amino Acid Incorporation Into Skeletal Muscle Proteins For the measurements of amino acid incorporation into skeletal muscle protein of sham-operated and septic rats (protein synthesis), muscle biopsies were taken from the medial aspect of quadriceps muscle under light either anesthesia. The muscle biopsy specimens were carefully dissected into fine strands of fibers, which were incubated in 1 mL of an incubation medium as described previously.6 To each incubation flask, 0.25 urn Ci L-[‘4C]tyrosine was added. All incubations were run in duplicate at 37°C in a shaking water bath at 100 to 110 oscillations per minute. Incubations were terminated after 2 hours by the addition of 1 mL of 10% (wthrol) trichloracetic acid (TCA) and the tissue was

homogenized using a Polytron homogenizer PCU-2 at position 6 for 20 seconds at 0°C; then centrifuged at 1,300 x g for 10 minutes. The precipitate was washed three times in ice-cold 5% (wt/vol) TCA and thereafter resuspended and heated at 90°C for 20 minutes in 5% (wt/vol) TCA. The precipitate was extracted in ether:ethanol(1:l) for 20 minutes and in ether for 20 minutes. The precipitate was dried at room temperature and dissolved in 1N NaOH. Ahquotes were taken for protein analysis and radioactivity was monitored by liquid scintillation spectrometry (LKB 1211 Rack-Beta scintillation counter, Wallacoy, Turku-10, Finland). The amount of tyrosine incorporated into TCA-precipitated proteins was determined from the specific radioactivity in the incubation medium. Rate of protein synthesis is lower in vitro than in vivo, but good agreement was found when qualitative changes in protein synthesis were measured both in vitro and in vivo in skeletal muscle during different conditions.” Degradation of Skeletal Muscle Proteins Rate of protein degradation was determined by measuring the amount of tyrosine released from incubated muscle tissue into the medium as described by Fulks et aL3s Since tyrosine is neither synthesized nor degraded in muscle, and since the intracellular pool of tyrosine remains constant during such incubations, the appearance of tyrosine in the medium provides an accurate estimate of protein degradation.“” For the measurement of protein degradation in muscles of sham-operated and septic rats, muscle biopsies (200 to 300 mg wet weight) were isolated as described above. The biopsies were washed in ice-cold Krebs-Ringer bicarbonate buffer (pH 7.4) for 15 minutes and then incubated at 37°C in 1.5 mL of the same buffer saturated with O&O, (191, vol/vol). Puromycin (200 &mL) was added to the incubation medium to prevent reincorporation of amino acids. Incubations were terminated after 2 hours as described above, and tyrosine concentration in the medium was determined as described previously.6 In preliminary experiments, the release of tyrosine into the incubation medium was linear with time for 3 hours.

RESULTS

decrease in body weight of septic (11.49%) rats was similar to that of sham-operated controls (11.09%) (Table 1). Sepsis resulted in an increase in rectal temperature of rats 48 hours after cecal ligation (+ 1.9’C). Sepsis resulted in increases in heart rate (28.5%, P < .Ol) and cardiac output (30.6%, P < .Ol), but with no marked changes in mean arterial pressure (Table 1). The urinary nitrogen excretion rate was increased by more than 63% in septic rats. Both sham-operated and septic rats were in a negative nitrogen balance (Table 1). Blood concentrations of pyruvate (145%), lactate (106%) glutamine (27.4%), and alanine (156%) in septic rats were higher than that found in corresponding controls (Table 1). Blood ketone bodies (61%) and plasma cholesterol (32.5%) concentrations in septic rats were lower than those of sham-operated rats (Table 1). No marked changes in the concentrations of blood glucose, nonesterified fatty acids, and tricylglycerols were observed in septic or shamoperated rats. Both plasma insulin and glucagon concentrations were increased by 2.2- and 2.4-fold in septic rats, respectively (Table 1). The results in Table 1 provide detailed information on the characteristics of septic and sham-operated rats used in the present work (see Discussion). The

Glutamine and Alanine Production by In Wro and In viva Hindlimb Preparations

In vitro preparations of skeletal muscle, skin, and adipose tissue were used to assess the effect of sepsis on the contribution of these tissues to the production of glutamine and alanine in rat hindlimbs. Table 2 shows the rates of glutamine and alanine production in forelimb epitrochlearis muscles (which is considered to be representative of glutamine and alanine production by the hindlimb musclesW), skin slices, and pads of adipose tissue that were obtained from sham-operated and septic rats. In shamoperated rats, muscular glutamine production was ll- to 15-fold higher than that of skin and adipose tissue, whereas alanine production was approximately two to four times greater than that of skin and adipose tissue, respectively, confirming previous work.” Sepsis resulted in markedly enhanced rates of production of both glutamine and alanine only by skeletal muscle preparation, with no significant

ARDAWI AND MAJZOUB

156

Table 1. Body Weight, Rectal Temperature,

Hemodynamic Parameters, Nitrogen Balance, and Blood Concentrations

Lactate, Glutamine, Alanine, Ketone Bodies, Plasma Cholesterol, Triacylglycerols, Nonesterified

of Glucose, Pyruvate,

Fatty Acids, Insulin, and Glucagon for 4B-Hour

Septic and Corresponding Control Rats Animals Septic

Sham-Operated Initial

body weight(g)

205.6 % 13.9 (15)

202.6 * 11.1 (15)

Final body weight(g)

162.6 2 11.6 (15)

179.5 * 10.4 (15)

Rectal temperature (“C)

35.62 ? 0.62 (6)

37.52 ? 0.49 (6)”

Heart rate (beats/min)

375 + 12 (10)

462 + 26 (10)t

Mean arterial pressure (mm Hg) Cardiac output (mL/kg/min)

115r5(10)

124 + 6 (10)

310 + 14 (10)

405 2 22 (10)t

Nitrogen-balance (mg of N/d/l00 g body wt)

-31.56

Blood glucose (mmol/L)

+ 6.62 (6)

-51.70

4.35 + 0.24 (6)

Blood lactate (mmol/L)

+ 7.22 (6)$

3.91 ? 0.13 (6)

1.44 t 0.55 (10)

2.96 2 0.69 (IO)*

Blood pyruvate (mmol/L)

0.056 + 0.012 (6)

0.142 ? 0.009 (6)Z

Blood glutamine (mmol/L)

0.506 + 0.054 (10)

0.647 2 0.036 (10)t

Blood alanine (mmol/L)

0.124 r 0.016 (10)

0.316 t 0.07 (lo)*

Blood ketone bodies (mmol/L)

1.11 + 0.13 (IO)

Plasma cholesterol (mmol/L)

2.34 z 0.33 (IO)

1.56 + 0.26 (IO)*

Plasma triacylglycerols (mmol/L)

0.71 + 0.30 (10)

0.67 r 0.19 (10)

Plasma non-esterified fatty acids (mmol/L) Plasma insulin (pU/mL) Plasma glucagon (pg/mL)

0.55 * 0.11 (lo)*

0.64 2 0.14 (6)

0.75 ? 0.09 (6)

11.09 + 4.03 (10)

35.14 + 9.47 (lo)*

533 + 47 (10)

1766

NOTE. Values are presented as means + SD, with the number of animals used given in parentheses.

+

355 (lo)*

Rats were starved for 46 hours after

sham-operation or cecal ligation as described in the Experimental section. Statistical significance: lP < .05, tP < .Ol, SP < ,001.

changes by skin and adipose tissue preparations (Table 2). Assuming that the production of glutamine and alanine by hindlimb muscle is of the same order as that of epitrochlearis muscle and based on the results presented in Table 2, skin and adipose tissue preparations contributed not more than 4% to the total production of glutamine in shamoperated and septic rats (Table 3). However, skin preparations contributed 21.4 and 13.6% to the total production of alanine by hindlimb of sham-operated and septic rats, respectively, whereas that of adipose tissue preparations contributed only 1.8% to 4.9% (Table 3). Therefore, in the work described below, metabolic studies were confined only to skeletal muscle preparations. Metabolite and Amino Acid Concentrations in Freeze-Clamped Hindlimb Muscles

Since the above results suggest that sepsis enhances the formation and production of both glutamine and alanine by skeletal muscle, the concentrations of key metabolites that

participate in the biosynthetic pathways for glutamine and alanine were determined in freeze-clamped muscles of sham-operated and septic rats and are shown in Table 4. Muscle glutamine and glutamate concentrations were decreased, whereas that of ammonia increased by about 69% in septic rats. It is suggested that the activity of AMPdeaminase (EC 3.5.4.6) is increased, as indicated by the elevated levels of ammonia and ionosine monophosphate (IMP) in muscles of septic rats (Table 4). The concentrations of AMP were decreased by approximately 21% (P < .OS),while that of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) were unchanged, in muscles of septic rats as compared with corresponding controls (Table 4). Calculation of the energy charge ratio ([ATP + 0.5 ADP]/[ATP + ADP + AMP]) showed no marked difference in muscles obtained from septic (0.929) or sham-operated (0.918) rats, respectively. The concentrations of pyruvate and 2-oxoglutarate (two oxoacids involved in glutamine and alanine biosynthesis) were increased

Table 2. Effect of Sepsis on the Rate of Production of Glutamine and Alanine by Skeletal Muscle, Skin, and Adipose Tissue Preparations for 4B-Hour Septic and Corresponding Control Rats Rata of Production

Condition of Tissue

Animals

Muscle

Sham-operated

Skin

Sham-operated

(5)

Septic (7) (6)

Septic (7) Adipose tissue

Sham-operated

(5)

Septic (7) NOTE. Tissues were isolated from sham-operated

I~mollhig

wet wtj

Glutamine

Alanine

2.63 2 0.32

2.06 it 0.16

4.57 f 0.55f

3.42 + 0.46’

0.24 -t 0.09

0.97 r 0.33

0.39 -t 0.07

1.32 2 0.39

0.19 * 0.06

0.52 2 0.11

0.19 ?z 0.05

0.46 + 0.12

or septic rats and incubated as described in the Experimental section. Rates are given as

means ? SD, with the number of animals used given in parentheses. Significance of differences from sham-operation values are indicated by lP < ,001.

MUSCLE GLUTAMINE METABOLISM

IN SEPSIS

159

Table 3. Contributions of Muscle, Skin, and Adipose Tissue to Glutamine and Alanine Production by the Hindlimb of Sham-Operated

TissueWeight (g/100 g wet WI of hindlimb)

Conditionof Animal

Tissue Muscle

Sham-operated

(5)

Septic (7) Skin

Sham-operated

(5)

Septic (7) Adipose tissue

Sham-operated

and Septic Rats

(5)

Septic (7)

CalculatedHindlimbProduction (~mol/h/lOOg wet wt of limb) Glutamine

Alanine

54.30 2 1.85

153

111

59.41 2 1.82

271

203

24.49 -t 1.22

5.9

23.8

20.95 2 2.61

8.2

27.7

10.41 2 1.06

2.1

5.4

8.17 + 0.90

1.5

3.7

NOTE. Constituents of hindlimbs (muscle, skin and adipose tissue) of sham-operated and septic rats were determined as described by Ruderman et aL6’ Total hindlimb production of glutamine and alanine was calculated using the values in Table 2, assuming forelimb epitrochlearis muscles to be a satisfactory representative of glutamine and alanine production by the hindlimb muscles. Results are given as means + SD, with the numbers of animals used shown in parentheses.

(38.7%, P < .OS) and decreased (30.6%, P < .OS) in response to sepsis, respectively (Table 4). The decrease in muscular glutamine concentration in septic rats was associated with changes in other amino acids. The following amino acids were decreased in muscles of septic rats as compared with corresponding controls: aspartate (18.2%), asparagine (18.2%) proline (44.4%), ornithine (18.9%), lysine (39.0%) histidine (24.6%), and arginine (34.8%) respectively (Table 5). However, the following amino acids were not markedly changed in response to sepsis: phosphoserine, taurine, threonine, serine, glycine, alanine, citrulline, cysteine, methionine, isoleucine, and phenylalanine (Table 5) whereas valine (52.5%) leucine (28.4%), and tyrosine (16.9%) were markedly increased in muscles of septic rats, respectively (Table 5). Plasma Amino Acids The total plasma amino acid pool was not significantly changed in septic rats (3.45 mmol/L in sham-operated, 3.48 mmol/L in septic rats). When individual amino acids were examined, several amino acids exhibited an increase, whereas others showed either a decrease or no change (Table 5). Plasma concentrations of taurine, asparagine, glutamate, proline, citrulline, methionine, and phenylalanine were increased in septic rats (Table 5) whereas those

Table 4. Metabolite Concentrations

of threonine, serine, glycine, valine, isoleucine, omithine, and arginine were decreased.

leucine,

Blood Flow and Arteriovenous Difference Measurements of Hindlimbs of Septic and Sham-Operated Rats

Arteriovenous concentration difference measurements of glutamine, alanine, glutamate, and ammonia across the hindlimbs of sham-operated and septic rats are presented in Table 6. Septic rats exhibited a marked increase in net release of both glutamine (63.8%) and alanine (46.7%), accompanied by an uptake of glutamate and ammonia by the hindlimbs as compared with that of sham-operated rats (Table 6). Hindlimb blood flow was decreased from 3.42 f 0.30 to 2.95 2 0.18 mL/min/lOO g body weight in septic rats (not statistically significant). In hindlimbs of septic rats, the net release of glutamine and alanine was enhanced by about 41.2% and 26.5%, respectively (Table 6). MuscularActivities of Glutamine Synthetase and Glutaminase

One possible explanation for the increased rates of glutamine production by muscles from septic rats as compared with sham-operated controls, is increased activity of the enzyme glutamine synthetase. In the present work, only

in Hindlimb Muscle of Sham-Operated

and Septic Rats

MetaboliteConcentration(kmollg of tissue) Metabolite

Sham-OperatedRats

Septic Rats

Glutamine

1.45 + 0.18 (16)

0.76 + 0.20 (16)$

Glutamate

1.88 2 0.43 (16)

0.86 + 0.16 (16)s

Alanine

1.61 f 0.11 (7)

1.54 + 0.11 (10)

Ammonia

0.32 + 0.08 (10)

0.54 r 0.09 (14)t

Pyruvate

0.049 t 0.013 (8)

0.068 * 0.012 (S)*

2-Oxoglutarate

0.049 + 0.006 (7)

0.034 + 0.009 (lo)*

ATP

4.49 ? 0.64 (5)

5.17 -t 0.25 (8)

ADP

0.75 + 0.07 (8)

0.74 f 0.04 (11)

AMP

0.062 + 0.010 (8)

0.049 2 0.022 (9)*

IMP

0.063 + 0.012 (8)

NOTE. Freeze-clamping

of skeletal muscles of hindlimb and measurements

0.135 ? 0.035 (8) t of metabolites were carried out as described in the Experimental

section. Values are presented as means 2 SD. Significance of differences from sham-operated control values are indicated by lP < .05, tP < .Ol, Sp < .Ol.

160

ARDAWI AND MAJZOUS

Table 5.

Concentration

of Plasma and Muscle Amino Acid Groups of Sham-operated Concentration

in Arterial

Concentration

Plasma (nmol/L) Septic

In = 241

Phosphoserine Taurine

in Hindlimb

Muscle lnmolig wet wt)

Sham-Operated Amino Acid

and Septic Rats

Sham-Operated

(n = 24)

24 2 5

25 + 5

241 k 36

292 c 76*

Septic

(n = 24)

(n = 24)

17 + 7

18 2 6

16,690 k 912

17,236 f. 184

Aspattate

17 k 9

17 -c 3

1,190 + 212

Threonine

200 lr 26

119 Ir 39s

2,167 c 88

2,068 -t 58

Serine

214 2 22

144 + 37s

2,329 + 94

2,161 -t 100

73 2 23’

347 k 33

284 k 57*

2,502 i 307

1,217 k 359*

Asparagine

59Ir

11

Glutamate

39 -c 9

75 k 8*

Proline

195 2 65

340 2 61*

755 k 108

Glycine

286 2 33

212 2 36’

17,185 2 294

16 2 3

9 ? 3s

973 + 254*

420 2 133* 17,487 + 463

402 k 15

391 k 13

193 2 36

137 -*- 22’

280 t- 36

427 k 76*

Cysteine

36 + 7

41 2 12

149 2 13

137 k 19

Methionine

49*

59 k 6*

84 5 25

92 2 28

121 + 25

138 -+ 31

Citrulline Valine

11

lsoleucine

102 + 29

83 + 11”

Leucine

171 2 39

122 + 36*

802

Tyrosine

14

692

11

88 2 28

113 + 36*

254 + 29

297 2 32* 80 k 30

71 2 13

91 k 18*

85 ? 42

Ornithine

111 ? 13

82 2 19*

74 + 30

Lysine

378 2 54

366 ? 33

1,564 + 220

Histidine

133 2 30

119 + 44

211 + 63

159 -t 43f

Arainine

154 + 45

122 + 52*

744 + 83

485 + 93*

Phenylalanine

60 + 27* 950 + 213*

NOTE. Values are means + SD, with n being the number of separate animals used. Significance of differences from sham-operated values, *P < .05.

the activity of glutamine synthetase of the quadriceps muscle of septic rats exhibited a marked increase as compared with sham-operated rats (Table 7). However, the activity of glutaminase was significantly decreased in muscles of septic rats (Table 7).

DISCUSSION

In the present work, the experimental animal model of sepsis used is considered to be a moderate form of sepsis compared with that of others, and septic rats were not hypothermic or shocked, confirming previous work.q.26Septic rats displayed similar physical symptoms of sepsis; namely a lack of active movement, piloerection, diarrhea, and a variable amount (usually 5 to 10 mL) of foul-smelling yellow-brown fluid contained in the peritoneal cavity, with multiple intraperitoneal abscesses. The microbial flora closely approximate that of human disease and therefore indicate that this animal model of sepsis is a suitable model of human disease.25 The concentration of blood lactate was increased in septic rats post operation as early as 12 hours (results not shown), but never reached values that would be classified as lactic acidosis (5 mmol/L); this confirms previous worky.‘6 with a similar form of sepsis. Hypoglycemia did not develop

Tyrosine Release and Incorporation Into Skeletal Muscle Proteins The extent of tyrosine released into the incubation medium of muscle preparations isolated from septic and sham-operated rats is shown in Table 8. Protein degradation of skeletal muscle proteins was increased by 82.3% in response to sepsis as compared with sham-operated rats. The extent of tyrosine incorporated into muscle proteins of septic and sham-operated rats is presented in Table 8. There was a marked decrease in the rate of incorporation of tyrosine in muscles obtained from septic (27.8%, P < .OS) compared with sham-operated rats (Table 8).

Table 6.

Blood Flow, Arteriovenous

Concentration

Differences, and Net Rates of Exchange for Glutamine, Alanine, Glutamate,

and Ammonia of Sham-Operated Arteriovenous

Condition of Animals

Sham-operated Septic

Hindlimb lmUmin/lOO

3.42 + 0.30 2.95 k 0.18

and Septic Rats

Difference

Hindlimb

(nmol/L)

Blood Flow Q bodv wtl

Concentration

Glutamine

-149 -244

+ 79 + 94t

Alanine

-107 -157

Exchange Rate

(nmollmin/100 Glutamate

Ammonia

Alanina

g body wt) Glutamate

Ammonu

-c 31

11 k3

82 k 27

-510

k 24

-366

+ 10

38 2 2

280 t 9

+ 23’

21 * 7t

90+

-720

?z 78t

-463

+ 20’

62 ? 13t

266 2 35

16

Glutamine

NOTE. Measurements and calculations were carried out as described in the Experimental section; results are presented as means + SDfor 10 to 12 rats in each group. Negative sign indicates release. Significant differences from sham-operated values are indicated by lP < .05, tP

< ,001.

MUSCLE GLUTAMINE METABOLISM

Table 7.

IN SEPSIS

161

Effect of Sepsis on the Activities of Glutamine Synthetase and Glutaminase in Various Skeletal Muscles of Sham-Operated Conditionof Animals

Soleus

Sham-operated

nmollmin/g

(6)

Septic (6) Gastrocnemius

Sham-operated

(6)

Septic (6) Quadriceps

Sham-operated

and Septic Rats

GlutamineSynthetaseActivity

(6)

Septic (6)

wet wt

nmollmin/mg

Glutaminase protein

nmollmidg

wet WI

Activity nmol/min/mg

protein

278 + 45

2.55 + 0.25

890 k 19

6.96 -c 0.15

277 + 40

2.56 + 0.26

795 k 54

4.73 k 0.33*

245 + 19

2.49 2 0.09

1,702 2 129

17.29 + 0.17

225 + 49

2.37 + 0.40

1,400 f 103*

14.74 f 0.12*

304 f 150

3.11 + 1.44

876 k 20

5.41 2 0.18

496 + 76’

5.20 + 0.28t

606 * 47s

3.79 f 0.29*

NOTE. The results are presented as means + SD, with the number of animals used given in parentheses. Values that are significantly different from sham-operated are indicated by lP < .05, tP < .Ol, W < ,001.

in septic rats of the present work, indicating that animals did not go through the late hyperdynamic septic state as described by Chaudry et a1.4’ Hyperglycemia and glucose intolerance are frequent manifestations of the metabolic response to sepsis,‘,42.43 while the plasma levels of insulin are normal or increased.“.45 In the present work, there was no change in blood glucose level in septic rats, but the plasma insulin was increased (Table 1). This suggests that insulin resistance is present in septic rats as found by others.’ However, it can be argued that the near threefold increase in plasma glucagon in septic rats (see Table 1) was sufficient to enhance gluconeogenesis and thus to maintain blood glucose; the latter is not possible since hepatic and renal gluconeogenesis were found to be impaired in similar septic rats.9,47Moreover, experiments on incubated soleus muscle obtained from septic rats demonstrated that there were no changes in the sensitivities of glycolysis or glycogen synthesis to insulin.” It is therefore possible that in vivo insulin resistance is caused by elevated levels of fatty acids operating through the glucose-fatty acid cycle. Several studies have shown increased plasma levels of stress hormones in stress that would cause increased plasma levels of fatty acids.3 However, in the present work, there was no change in the plasma level of fatty acids (Table 1) inspite of a threefold increase in plasma insulin; the latter suggests insulin resistance with regards to lipolysis. Further work is needed in this respect. In the present work, plasma glutamine and alanine were increased with total plasma amino acids remaining unchanged (see Table 5), inspite of accelerated proteolysis in skeletal muscle (Table 8). The changes in the concentration of various plasma amino acids are consistent with previous reports in patients and experimental animals with sepsis.4S*a.49 The phenylalanine/tyrosine ratio in plasma was previously demonstrated to reflect the degree of inflammation and catabolic state during infection and an increased Table 8. Rates of Tyrosine Incorporated

ratio was suggested to reflect accelerated skeletal muscle catabolism?’ In the present work, this ratio was increased from 0.89 to 1.32 in septic rats as compared with shamoperated controls. This suggests that the increased phenylalanine/tyrosine ratio not only reflects accelerated protein breakdown in skeletal muscle, but might also reflect changes in hepatic amino acid uptake and metabolism. Further work is needed to support the latter suggestion. In the present work, skeletal muscles of the hindlimb contributed approximately 87.3% and 47.9% of the total calculated production of glutamine and alanine in response to sepsis, respectively (Table 3). Unlike the total plasma amino acid nitrogen, which remained stable, intracellular free amino acid nitrogen concentrations of skeletal muscle of septic rats was decreased with approximately 49% of the nitrogen loss accounted for by the decrease in glutamine concentration. In addition, intracellular alanine concentration in muscle was increased in response to sepsis. Similar changes in skeletal muscle free amino acid concentrations have been described in catabolic patients and experimental animals.4,5,23,29.48.51 Previous work by Ardawi has shown accelerated rates of glutamine production and release both in vitro and in vivo by skeletal muscles of thermally injured rats.29 Moreover, it has been demonstrated that glutamine release and/or production by skeletal muscle preparations are markedly accelerated after surgery, uremia, uncontrolled diabetes, and sepsis.7,8V’7,‘8 The present work showed similar changes in glutamine production by skeletal muscle of septic rats. Four lines of evidence to support the latter view: (1) rates of production of glutamine from in vitro muscle preparations (Table 2); (2) blood flow plus arteriovenous concentration difference measurements across the hindlimb of the rat (Table 6); (3) changes in the concentration of metabolites involved in glutamine biosynthesis (Table 4); and (4) changes in vitro in the maximum catalytic activity of glutamine synthetase (a key enzyme in the biosynthesis of glutamine) (Table 7).

Into Muscle Proteins and Released From Incubated Muscle Tissue of Septic and Sham-Operated Tyrosina

Animals

Sham-operated Septic

Incorporation

(nmol/h/g

protein)

115+37(6) 83 + 19 (6)’

Rats

Tyrosine Release

(wmol/h/gprotein) 23.40 2 3.21 (7) 42.66 + 7.20 (7)t

NOTE. Isolation and incubation of muscles together with tyrosine incorporation or release were carried out as described in the Experimental section. Values are presented as means 2 SD. Significance of differences from sham-operated values, lP < .05, tP < .OOl.

162

ARDAWI AND MAJZOUB

Several factors could have contributed to the diminished intracellular glutamine concentration in skeletal muscles of septic rats: (1) an inhibition of uptake and/or accelerated release of glutamine in muscle due to changes in the transport system properties; (2) decreased synthesis; and (3) increased degradation of glutamine in muscle. Glutamine has been found to be transported by a specific carrier system in rat muscle, which is noncompetitively inhibited by leucine.” It is possible, therefore, that the changed plasma amino acid pattern in response to sepsis (see Table 6) may result in changes in the concentrations of amino acids competing with glutamine for the transport system. However, this was not the case as far as leucine is concerned, since its plasma levels decreased in response to sepsis. Moreover, it is possible that the diminished muscle glutamine in septic rats could be related to a change in the ability of the glutamine transporter to maintain the concentration gradient across muscle. The latter is Na’dependent,52 and so the factors that influence Na’ transport, either by changing the activity of the Na+/K+-ATPase or by influencing membrane permeability to Na+, could influence the glutamine gradient. One factor is insulin, which directly stimulates Na’ transport.5”54 Because insulin also regulates protein synthesis at the level of translation,55 the apparent insulin resistance that was evident in septic rats could result in altered muscle Na’ transport, with concomitant changes in the glutamine concentration and protein synthesis. The relevance of this is that the inhibition of muscle protein synthesis in endotoxaemia is correlated with a decrease in the concentration of muscle glutamine, and in a perfused rat hindlimb preparation in which glutamine concentrations are directly related to the rate of protein synthesis.57 The decrease in muscle glutamine concentration may also contribute to the accelerated protein degradation rate, because this amino acid inhibits degradation in cultured skeletal muscle cells.58 In our experiments, the inhibition of protein synthesis (as compared with sham-operated-controls, Table 8) and accelerated rates of protein degradation (as indicated by muscle tyrosine release), were associated with a reduction in glutamine concentration, thus confirming previous findings (see above). Therefore, impaired insulin action with sepsis is one possible link between the diminished glutamine concentration and possible changes in protein synthesis. Similar conclusions were suggested in endotoxemic and

corticosteroid-treated rats.56.5u.mAnother factor could be the direct effect of the endotoxin or associated cytokines on ionic balance or flux across muscle.6’,62Furthermore, it is possible that endogenous sources of glutamine (there is no exogenous source of glutamine for animals used in the present work since animals were starved) may not be sufficient to prevent the muscular depletion of glutamine in response to sepsis; the latter could be related to decreased capacity of available glutamine carrier systems to transport the amino acid across cell membranes. Further work is needed to support the latter suggestion. Results obtained from arteriovenous concentration difference measurements across the hindlimbs of septic rats (Table 4) showed a significant increase in the efflux of glutamine as compared with that of sham-operated rats. This was inspite of the declining concentrations of muscular glutamine. These findings suggest that sepsis decreased and/or increased the influx and efflux of muscular glutamine, respectively. Finally, the enhanced efflux of glutamine from the hindlimb could not have originated entirely from muscle protein. This is indicated by the finding that glutamine contributes a minor percentage of skeletal muscle proteins.” Thus, an increase in net glutamine synthesis must have occurred in response to sepsis. The latter is consistent with the changes in muscular glutamate, 2-oxoglutarate, and ATP concentrations, which were accompanied by enhanced glutamine synthetase activity (Tables 4 and 7). The results of the present work parallel, in many respects, alterations that occur in septic patients.“.lb Specifically, there is a similar decrease in glutamine concentration and thus in total free amino acid nitrogen in skeletal muscle, which is associated with enhanced amino acid efflux from skeletal muscle and negative nitrogen balance. Clearly, more work is needed to characterize both the exact nature of the association between muscular glutamine and protein synthesis and/or degradation and the factors that may contribute to the changes in muscular glutamine concentrations. Increased understanding of such phenomena would help in the development of therapies directed at correcting the decrease in muscle glutamine during catabolic conditions.

ACKNOWLEDGMENT We thank

Nimira

Mediratta

for excellent

secretarial

assistance.

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