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De novo glutamine synthesis induced by corticosteroids in vivo in rats is secondary to weight loss
ARTICLE IN PRESS Clinical Nutrition (2004) 23, 1035–1042
www.elsevier.com/locate/clnu
ORIGINAL ARTICLE
De novo glutamine synthesis induced by corticosteroids in vivo in rats is secondary to weight loss Ivo de Blaauwa,*, Annemie M.W.J. Scholsb, Esther Koerts-deLangb, Emiel.F.M. Woutersb, Nicolaas E.P. Deutza a
Department of Surgery, Maastricht University, Maastricht, The Netherlands Department of Pulmonology, Maastricht University, Maastricht, The Netherlands
b
Received 8 January 2003; accepted 9 January 2004
KEYWORDS Glutamine; Protein turnover; Muscle; Stress; Glucocorticosteroids; Rats
Summary Introduction: Corticosteroid treatment affects muscle protein and glutamine metabolism. In the present study we aimed to clarify to what extent anorexia, weight loss and corticosteroids determine protein and glutamine metabolism in muscle. Methods: The study was performed in Wistar rats (300–350 g, n ¼ 40) given triamcinolone (0.25 mg/kg/day i.m.) treatment (CS group) for 14 days, sham treated free fed (FF group), sham treated pair fed (PF group) and sham treated pair weight (PW group). In vivo protein and glutamine turnover were measured using L-[2,63H]phenylalanine and L-[3,4-3H]glurtamine as tracer in a three compartment model across the hindquarter. Results: Corticosteroid treatment decreased total body weight to a greater extent than can be explained by decreased food intake only, justifying the need for pair weight controls. Muscle weight loss was relatively greater in the corticosteroid treated rats than in the pair weight controls indicating specific corticosteroid induced changes in muscle protein metabolism. Pair weight rats increased muscle net protein breakdown rates from 573 nmol 100 g body weight1 min1 to 1573 nmol 100 g body weight1 min1 (Po0:05 vs FF). In the corticosteroid treated rats net protein breakdown rates increased to 2274 nmol 100 g body weight1 min1 (Po0:01 CS vs FF/PF) Net protein breakdown in corticosteroid treated rats was accompanied by increased glutamine efflux from the hindquarter (Po0:05; CS vs FF/PF/PW). The latter could predominantly be explained by de novo synthesis. Furthermore, corticosteroid treatment induced a loss of plasma to free muscle glutamine gradient indicating down regulation of glutamine membrane transport rates into muscle. This effect was, however, similar in the pair weight control group and can thus be fully accounted for by the muscle weight loss. Conclusion: Two weeks treatment with triamcinolone increases net in vivo protein breakdown of muscle directly and indirectly due to secondary weight loss and decreased food intake. The amino acid residues are used for glutamine de novo
*Corresponding author. Department of Pediatric Surgery, UMC St. Radboud Hospital, PO box 9101, HP 413, 6500 HB Nijmegen, The Netherlands. Tel.: þ 31-24-3619761; fax: þ 31-24-3613547. E-mail address: [email protected] (I. de Blaauw). S0261-5614/$ - see front matter & 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.clnu.2004.01.004
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synthesis which is exported from muscle to visceral organs by down regulation of glutamine transport systems. These changes were in majority related to muscle weight loss. & 2004 Elsevier Ltd. All rights reserved.
Introduction Glucocorticoids have a prominent role in the catabolic response to sepsis, trauma and cancer. Chronic use of glucocorticoids in patients with inflammatory disease, e.g. Crohn’s disease, rheumatoid arthritis or chronic obstructive pulmonary disease, may also results in weight loss and muscle catabolism.1,2 In vitro, glucocorticoids induce muscle atrophy by increasing protein breakdown rates through activating the ATP-Ubiquitin-dependent pathways.3 In vivo, decreased protein synthesis rates have also been observed,4 but protein breakdown and synthesis have never been measured in vivo simultaneously. Furthermore, in previous experimental studies control groups were normally fed. Anorexia is one of the side effects of corticosteroid treatment and increased corticosteroid plasma levels.5 Anorexia can explain part of the muscle wasting. Therefore, pair fed controls should always be included. However, corticosteroids induce a greater weight loss than can be explained by anorexia only. Weight loss itself also induces changes in protein and amino acid metabolism of muscle. In vivo measurements of effects of chronic corticosteroid usage are thus measurements of direct corticosteroids effects but also indirect effects of weight loss induced by previous corticosteroid usage.6 Therefore, effects of corticosteroids should not only be compared with pair fed but also pair weight controls. Net protein breakdown of muscle induced by corticosteroids results in production of amino acids. These amino acids are taken up by visceral organs for gluconeogenesis and acute phase protein synthesis.7 The amino acid glutamine is consumed by visceral organs in the acute and chronic phase of inflammation not only for gluconeogenesis and protein synthesis but also for acid base homeostasis, glutathione synthesis and as energy substrate.7 The majority of glutamine is produced by hindquarter muscle. Recently, we described a model to measure in vivo glutamine consumption and production in muscle.8 Within this model it is also possible to determine in vivo de novo production. Our aim in the present study was to study in vivo changes in protein synthesis and breakdown rates of muscle induced by corticosteroids. We further aimed to study changes in in vivo muscle
glutamine metabolism induced by corticosteroids. The study was also conducted to clarify to what extent metabolic changes in muscle are caused by corticosteroids per se or by related food deprivation or weight loss.
Methods Animals Male Wistar rats (300–350 g, Harlan-Winkelmann, Borchen, Germany) were individually housed in metabolic cages during the experiment after an adaptation period of 3 days. Rats were given standard laboratory rat chow and subjected to standard 12 h light–dark cycle periods (7:30 A.M.– 7:30 P.M.) with room temperature maintained at 251C. The experiments were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals9 and approved by the Ethical Committee of Animal Research of the Maastricht University.
Experimental groups Rats (n ¼ 40) were randomly divided into four groups. 1. 2. 3. 4.
Free fed control group. Pair fed control group. Pair weight control group. Corticosteroid group.
The corticosteroid group received a daily injection of triamcinolone of 0.25 mg/kg body weight (1.25 mg/ml) intramuscular for 2 weeks. Triamcinolone is a fluorinated corticosteroid with increased anti-inflammatory potency. The dose given in the present study has previously been used as model to describe corticosteroid induced muscle atrophy and disturbances in energy metabolism.5 Food intake in the corticosteroid group was ad libitum. The free fed control group had food intake ad libitum. The pair fed group had equal food intake as the corticosteroid group. The pair weight group had comparable body weight as the corticosteroid group by adjusting daily food intake. All control groups received daily injections of 0.05 ml
ARTICLE IN PRESS Muscle glutamine synthesis and corticosteroids
NaCl (150 mM) intramuscular for 2 weeks. All injections were given in the hamstrings of the left leg.
Study procedures Rats were randomly assigned to metabolic cages, taking an adaptation period of 4 days prior to the experiment into account. Hindquarter amino acid fluxes, protein and glutamine turnover were examined as described previously.8,10,11 In brief, postabsorptive, under ether anesthesia and at constant, pre-anesthesia body temperature, rats were subjected to a laparotomy. The right renal vein, the inferior caval vein and aorta were cannulated using a 25 gauge needle fixed in a Silastic tube (Silastic Medical Grade tubing 0.051 cm ID, 0.094 OD, Dow Corning Corporation, Midland, MO, USA) and fixed with cyano-acrylate. For flow measurements, the indicator dilution method with para-aminohippuric acid (PAH) was used (pH 7.4, iso-osmolaric, Sigma A 1422, St Louis, MI, USA). A primed constant infusion (0.75 ml 100 g bw1 h1, 5 mmol/l) of PAH was infused in the aorta, using a Minipuls 3 Peristaltic Pump (Gilson Medical Electronics Inc., Villiers-le-Bel, France). For protein and glutamine turnover studies, a primed constant infusion of L-[2,6-3H]phenylalanine and of L-[3,4-3H]Glutamine (NEN Dupont, NET-493, Mechelen, Belgium) was given in the right renal vein. 3H-Phenylalanine and 3 H-glutamine was given at a rate of 1 mCi 100 g bw1 h1. A minimum infusion time of 30 min was needed to reach steady state concentrations for PAH, 3H-phenylalanine and 3H-glutamine.11 The right carotid artery was cannulated during the equilibrium period with PE 50 catheter (Intramedic, 0.051 cm ID, 0.094 cm OD, Clay Adams, Parispany, NY, USA). The complete procedure took approximately 40 min. Blood (1.0 ml per catheter) was simultaneously sampled from the caval vein and carotid artery. All blood was collected in heparinized cups on ice. Hereafter, the right gastrocnemius muscle was dissected free, freezeclamped, put in liquid nitrogen and stored at 801C until further analysis. Gastrocnemius muscle from the left leg was prepared and weighted. For PAH determinations, 50 ml heparinized blood was added to 500 ml TCA 10%, vortexed and centrifuged at 8900g at 41C. The supernatant was frozen in liquid nitrogen and stored at 801C until further analysis. For hematocrit determinations a microhematocrit tube was filled with heparinized blood and centrifuged at 10,000g at room temperature. Hematocrit was read with a Micro Hematocrit
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Reader. Plasma was obtained by whole blood centrifugation at 8900g at 41C for 5 min. For ammonia determinations, 200 ml plasma was vortexed with 20 ml TCA 50%, put into liquid nitrogen and stored at 801C. To determine plasma amino acid specific activities and concentrations, 200 ml plasma was added to 8 mg 5-sulfosalicylic acid, vortexed, frozen in liquid nitrogen and stored at 801C. For the determination of tissue amino acid concentrations and specific activities, tissue was pulverized using a mortar and pestle precooled in liquid nitrogen. The tissue was further homogenized and deproteinized in a Mini-Beadbeater. Approximately 100 mg tissue was added to 400 ml SSA 5%, with 300 mg glass beads (diameter 1 mm), and beaten for 30 s. The homogenate was centrifuged at 41C at 11,000g and the supernatant frozen in liquid nitrogen and stored at 801C until further determinations. To determine tissue dry weight and water content approximately 200 mg pulverized tissue was freeze dried for 24 h in a Speedvac connected with a refrigerated condensation trap (Savant Instruments Inc., Farmingdale NY, USA).
Biochemical analysis Plasma ammonia and PAH were determined spectrophotometrically on a Cobas Mira S by standard enzymatic methods, using commercially available kits.10,11,8 Plasma and tissue amino acid concentrations and specific activity were determined by fully automated HPLC as described previously.12
Calculations Plasma flow across the hindquarter was calculated using PAH in the indicator dilution method.10,11,8 Substrate fluxes are calculated by multiplying the venous-arterial concentration differences with the mean plasma flow of the group and are expressed in nmol 100 g body weight1 min1. A positive flux indicates net release, a negative flux reflects a net uptake. Branched chain amino acids (BCAA) are the sum of valine, leucine and isoleucine. Total release of a-amino-nitrogen was calculated as the sum of the individual amino acids which are released by the hindquarter except taurine, arginine and alanine.12 Tissue concentrations of amino acids are expressed in mmol/l and derived by dividing the tissue homogenate amino acid concentration (mmol/kg wet weight) with the tissue water content. Whole body protein and glutamine turnover were determined by calculating the arterial dilution of tracer amino acid at steady state.13 Protein turnover rates were calculated by the A-V
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dilution of tritium labeled phenylalanine across the hindquarter calculated in a three compartment model.8,14,15 Intracellular disposal (D) and production (P) of phenylalanine can thus be calculated. Production of phenylalanine reflects protein breakdown and disposal reflects protein synthesis because muscle metabolism of phenylalanine is restricted to synthesis into and breakdown from protein.16 Intracellular production and disposal of glutamine were calculated using the same model.14,8 Knowing the relative amino acid concentration of phenylalanine and glutamine in muscle protein enables us to calculate glutamine production derived from protein breakdown and de novo synthesis.8,17
Statistical analysis Results are presented as mean7SEM. Statistical analysis was performed using the statistical package SPSS.18 Significance between groups was tested non-parametrically by Kruskal-Wallis test. Significance was considered present at Po0:05:
Results Food intake is depicted in Fig. 1. Corticosteroid treatment reduced food intake in the first five days.
Time (days) Figure 1 Food intake of different groups during the 2 week experiment; free fed (J), pair fed (n), pair weight (m) and corticoid treated rats (K).
Table 1
Food intake gradually returned to control values hence after. Body weight (Table 1, Fig. 2) decreased in the corticosteroid rats by 20% of the initial bodyweight. Bodyweight of the pair weight rats decreased similarly by 22%. Both control and pair fed rats had increased body weights. Gastrocnemius dry weights decreased both in the corticosteroid group and the pair weight group (Table 1). However, gastrocnemius weight expressed as percentage of body weight, was only significantly decreased in the corticosteroid group. Arterial ammonia decreased in both pair weight and corticosteroid treated rats (Table 2). Arterial phenylalanine only decreased in corticosteroid treated rats. Glutamine concentration remained unchanged. Total arterial amino acid concentration decreased in the pair weight and corticosteroid treated rats. Hindquarter plasma flow remained unchanged in all groups (Table 3). Ammonia efflux decreased in corticosteroid rats. Phenylalanine efflux increased in corticosteroid treated rats. Phenylalanine efflux increased to a similar extent in the pair weight rats indicating a similar net protein breakdown in both groups. Branched chain amino acid efflux from the hindquarter decreased in corticosteroid treated rats. Glutamine efflux increased in pair weight rats. In corticosteroid treated rats it increased significantly further (Po0:05 vs PW). Total a-amino nitrogen efflux increased in both pair fed and pair weight rats and increased most in corticosteroid treated rats. Intramuscular concentrations are depicted in Table 4. Intracellular ammonia decreased most in corticosteroid treated rats. Glutamine concentrations increased in the pair weight rats but not in the corticosteroid treated rats. Whole body protein turnover, given as Rate of Appearance, remained unchanged (Table 5). No significant changes were observed in hindquarter protein synthesis (disposal) and hindquarter protein breakdown. The difference (net balance), however, did increase in the control weight and corticosteroid treated rats. Whole body glutamine turnover remained unchanged in all groups (Table 6). Glutamine disposal by hindquarter muscle remained unchanged. Glutamine production
Body weight and gastrocnemius weight. FF
Total body weight Weight gastrocnemius Gastrocnemius/bodyweight
34377 1.8970.05 0.5670.01
PF 33473 1.8870.03 0.5670.01
PW
CS b
26674 1.5570.02b 0.5870.01
25175b,d,e 1.2670.05b,d,f 0.5070.01a,c,f
Values are mean7SEM. Body weight and gastrocnemius in grams. FF, free fed; PF, pair fed; PW, pair weight; CS, corticosteroid treated group. Kruskall-Wallis: a,bPo0:05; Po0:001 vs FF; c,dPo0:01; Po0:001 vs PF; e,fPo0:05; Po0:01 vs PW.
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by the hindquarter, however, increased in pair weight and corticosteroid treated rats. This was in majority an increase in de novo glutamine production. Glutamine net balance by hindquarter muscle increased in pair weight rats. In corticosteroid treated rats glutamine net balance increased significantly further (Po0:05 vs PW). Glutamine plasma-free muscle gradient is considered to be a function of membrane transport carriers.29,30 The plasma-free muscle gradient decreased to a similar extent in both pair weight and corticosteroid treated rats (Table 6).
Figure 2 Percentual weight change of different groups during the 2 week experiment; free fed (J), pair fed (n), pair weight (m) and corticoid treated rats (K).
Table 2
Arterial ammonia and amino acid concentrations.
Ammonia Phenylalanine BCAA Glutamine Glutamate Total a-amino nitrogen
FF
PF
PW
CS
34377 6973 404727 598720 8777 30027135
33473 6074 343729a 660742 7376 28227154
26674a 5872 358719a 652736 5672 2683769a
25175a 4372a 241715b,d,f 525720 108711 2007766b,d,f
Values are mean7SEM. Arterial concentrations in mmol/l. BCAA branched chain amino acids (valine, leucine, isoleucine). Kruskall-Wallis: a,bPo0:05; Po0:001 vs FF; c,dPo0:01; Po0:001 vs PF; e,fPo0:05; Po0:01 vs PW.
Table 3
Hindquarter blood flow, ammonia and amino acid fluxes.
Flow Ammonia Phenylalanine BCAA Glutamine Glutamate Total a-amino nitrogen
FF
PF
PW
CS
3.170.5 69720 574 72713 149798 1974 1547207
3.971.2 92727 1276 59721 1447128 2378 4847307
3.070.9 94729 1573 71711 3227130a 2073 4767215
4.070.8 16728a 2274b,d 13714a,h 5067140c,e,g 85718b,e,h 11277196c,e,g
Values are mean7SEM. Plasma flow in ml/100 g body weight/ min. Fluxes in nmol/100 g body weight/min. BCAA branched chain amino acids (valine, leucine, isoleucine). Kruskall-Wallis: a,b,cPo0:05; Po0:01; Po0:001 vs FF; d,e,fPo0:01; Po0:01; Po0:001 vs PF; g,hPo0:05; Po0:01 vs PW.
Table 4
Intramuscular ammonia and amino acid concentration.
Ammonia Phenylalanine BCAA Glutamine Glutamate Total a-amino nitrogen
FF
PF
PW
CS
1224779 6877 349718 2414751 685735 250227791
10427125 5273 317726 2517799 700770 248387361
876764b 5473 321721 3143799c 743769 255877570
397742c,f,i 4273b,e,g 251712b,d,g 24237190h 721763 226037556a,d,g
Values are mean7SEM. Intracellular concentrations are in mmol/kg wet weight. BCAA branched chain amino acids (valine, leucine, isoleucine). Kruskall-Wallis: a,b,cPo0:05; Po0:01; Po0:001 vs FF; d,e,fPo0:01; Po0:01; Po0:001 vs PF; g,h,iPo0:05; Po0:01; Po0:001 vs PW.
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Table 5 Whole body phenylalanine rate of appearance, hindquarter intracellular phenylalanine disposal, production and net balance.
Whole body rate Hindquarter Disposal Production Net Balance
FF
PF
PW
CS
595715
637744
606750
569722
2775 3375 574
2177 3375 1276
2175 3977 1573a
1575 3775 2274b,d
Values are mean7SEM. Data in nmol/100 gr body weight/min. Kruskall-Wallis: a,b,cPo0:05; Po0:01; Po0:001 vs FF; d,e,fPo0:01; Po0:01; Po0:001 vs PF;
g,h
Po0:05; Po0:01 vs PW.
Table 6 Whole body glutamine rate of appearance, hindquarter intracellular glutamine disposal, production, net balance and glutamine artery-muscle free ratio.
Whole body Rate Hindquarter Disposal Production De novo production Net balance Artery/free muscle ratio
FF
PF
PW
CS
51317426
55787442
60517471
55747336
246777 394761 329762 149798 0.2570.01
342782 4857118 4197110 1447128 0.2770.02
247734 5697112a 499796c 3227130a 0.2170.01a,c
302770 7297134a 6567100c 5067140c,e,g 0.2170.01a,c
Values are mean7SEM. Data in nmol/100 g body weight/min except glutamine artery/free muscle ratio. Kruskall-Wallis: a,b,cPo0:05; Po0:01; Po0:001 vs FF; d,e,fPo0:01; Po0:01; Po0:001 vs PF; g,hPo0:05; Po0:01 vs PW.
Discussion In the present study corticosteroid treatment increased weight loss to a higher extent than pair feeding. Pair weight controls are therefore the most appropriate controls. Corticosteroid treatment induced a relatively greater loss of muscle tissue than the muscle weight loss induced in pair weight controls. Steroids therefore did have specific catabolic effects on muscle. These include increased net protein breakdown of muscle and hindquarter glutamine efflux.
protein breakdown. Approximately 60% of this increase also occurred in the pair weight group. The muscle wasting after corticosteroid treatment is partly secondary to weight loss. Net protein breakdown of muscle resulted in a significant increased efflux of total amino acids by the hindquarter. In the corticosteroid group the increased efflux occurred in conjunction with decreased arterial total amino acid concentrations. This indicates that the clearance of amino acids from the arterial pool by visceral organs also increased.
Effects of corticosteroids on protein kinetics
Effects of corticosteroids on muscle glutamine kinetics
Corticosteroid induced muscle wasting has previously been associated with decreased protein synthesis rates.19 In other studies increased protein breakdown rates have been observed.3,20,21 All these studies, however, used free fed controls and measurements were mostly ex vivo. In our study protein synthesis and breakdown were simultaneously measured in vivo. We observed decreased protein synthesis and increased protein breakdown rates, although both not significantly. These subtle changes in protein synthesis and breakdown, however, did result in a significant increase in net
Approximately 30% of the amino acids released by hindquarter muscle is glutamine.22 This is confirmed in our study. Glutamine is considered a conditional essential amino acid in times of stress when plasma corticosteroid levels are elevated. Following corticosteroid treatment, interorgan glutamine flux is characterized by an increased glutamine efflux from muscle.23,24 We also observed an increased efflux after corticosteroid treatment in conjunction with increase in vivo glutamine production in muscle. In part glutamine production was derived from protein breakdown
ARTICLE IN PRESS Muscle glutamine synthesis and corticosteroids
but the majority came from de novo synthesis. This is indeed consistent with increased glutamine synthetase enzyme activity and mRNA expression.25–27 De novo glutamine synthesis also increased in the pair weight group. The increase is thus not only a direct effect of corticosteroids but can in part be explained secondary to the weight loss. Fasting and muscle weight loss is known to increases glutamine synthetase activity.28 Plasma-tissue glutamine gradient was decreased similarly in the pair weight group and the corticosteroid group. Maintenance of plasma tissue gradient is a function of glutamine membrane transport carriers. These carriers are suggested to be under direct influence of corticosteroids.29,30 Previous studies showed that corticosteroids down regulate System Nn inward directed membrane transport24 and thus increase glutamine efflux from muscle with a decreased plasma-tissue gradient. In our study similar effects on plasma-tissue glutamine gradient were seen in the pair weight control and corticosteroid treated rats and the effect of corticosteroids could thus be fully explained by the weight loss.
Effects of steroids on other amino acids Glutamate is one of the precursors for glutamine de novo synthesis in muscle. The observed increase in glutamate uptake by muscle can only explain a small part of the increased de novo synthesis. Branched chain amino acids can also be converted to glutamine by muscle. However, branched chain amino acid uptake decreased in the corticosteroid treated rats. Therefore, the major part of branched chain amino acids used for glutamine de novo synthesis had to be derived from protein breakdown. BCAA contribute to approximately 20% of the amino acid residues after protein breakdown.24 Decreased intracellular BCAA concentrations were observed despite increased protein breakdown. This indicates that the BCAA are converted to other amino acids, most probably glutamine. In previous studies increased BCAA dehydrogenase activity has been observed in response to corticosteroids.31 The majority of glutamine production by muscle can thus be explained by de novo production from BCAA derived from protein breakdown.
Conclusion Two weeks treatment with the synthetic corticosteroid triamcinolone increases in vivo net protein
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breakdown of muscle. The amino acid residues are in majority used for glutamine de novo synthesis. Down regulation of glutamine membrane transport rates into muscle cells resulted in an increased glutamine efflux of hindquarter muscle. This was mostly a secondary effect due to weight loss. Glutamine clearance by visceral organs also appeared to be up regulated in response to corticosteroids.
Acknowledgements The authors wish to thank Mr. HMH van Eijk and Mr. DR Rooyakkers for analytical help and Mrs GAM ten Have for biotechnical assistance.
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13. Wolfe RR. Tracers in metabolic research. New York: Alan R. Liss Inc; 1992. 14. Biolo G, Chinkes D, Zhang XJ, Wolfe RR. A new model to determine in vivo the relationship between amino acid transmembrane transport and protein kinetics in muscle. J Parenter Enteral Nutr 1992;16:305–15. 15. Biolo G, Fleming RY, Maggi SP, Wolfe RR. Transmembrane transport and intracellular kinetics of amino acids in human skeletal muscle. Am J Physiol 1995;268:E75–84. 16. Tourian A, Goddard J, Puck TT. Phenylalanine hydroxylase activity in mammalian cells. J Cell Physiol 1969;73:159–70. 17. Kominz DR, Hough A, Symonds P, Laki K. The amino acid composition of actin, myosin, tropomyosin and the meromyosins. Arch Biochem Biophys 1954;50:148–59. 18. Norusis MJ. SPSS/PC þ V4.0 BASE MANUAL for the IBM PC/ XT/AT and PS/2. Chicago, IL: SPSS Inc; 1989. 19. Millward DJ, Garlick PJ, Nnanyelugo DO, Waterlow JC. The relative importance of muscle protein synthesis and breakdown in the regulation of muscle mass. Biochem J 1976;156:185–8. 20. Tiao G, Fagan J, Roegner V, et al. Energy-ubiquitin-dependent muscle proteolysis during sepsis in rats is regulated by glucocorticoids. J Clin Invest 1996;97(2):339–48. 21. Kayali AG, Young VR, Goodman MN. Sensitivity of myofibrillar proteins to glucocorticoid-induced muscle proteolysis. Am J Physiol 1987;252:E621–6. 22. Ruderman NB, Berger M. The formation of glutamine and alanine in skeletal muscle. J Biol Chem 1974;249: 5500–6.
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23. Muhlbacher F, Kapadia CR, Colpoys MF, Smith RJ, Wilmore DW. Effects of glucocorticoids on glutamine metabolism in skeletal muscle. Am J Physiol 1984;247:E75–83. 24. Hundal HS, Babij P, Taylor PM, Watt PW, Rennie MJ. Effects of corticosteroid on the transport and metabolism of glutamine in rat skeletal muscle. Biochim Biophys Acta 1991;1092:376–83. 25. Abcouwer SF, Bode BP, Souba WW. Glucocorticoids regulate rat glutamine synthetase expression in a tissue-specific manner. J Surg Res 1995;59(1):59–65. 26. Max SR, Mill J, Mearow K, et al. Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles. Am J Physiol 1988;255:E397–402. 27. Minet-Quinard R, Moinard C, Villie F, et al. Kinetic impairment of nitrogen and muscle glutamine metabolisms in old glucocorticoid-treated rats. Am J Physiol 1999;276:E558–64. 28. King PA, Goldstein L, Newsholme EA. Glutamine synthetase activity of muscle in acidosis. Biochem J 15-11-1983; 216(2):523–5. 29. Millward DJ, Jepson MM, Omer A. Muscle glutamine concentration and protein turnover in vivo in malnutrition and in endotoxemia. Metabolism 1989;38:6–13. 30. Rennie MJ, Willhoft NM, Taylor PM. Glutamine transport and metabolism in mammalian skeletal muscle. Biochem J 1992;285:339–44. 31. Block KP, Richmond WB, Mehard WB, Buse MG. Glucocorticoid-mediated activation of muscle branched-chain alphaketo acid dehydrogenase in vivo. Am J Physiol 1987;252: E396–407.
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ORIGINAL ARTICLE
De novo glutamine synthesis induced by corticosteroids in vivo in rats is secondary to weight loss Ivo de Blaauwa,*, Annemie M.W.J. Scholsb, Esther Koerts-deLangb, Emiel.F.M. Woutersb, Nicolaas E.P. Deutza a
Department of Surgery, Maastricht University, Maastricht, The Netherlands Department of Pulmonology, Maastricht University, Maastricht, The Netherlands
b
Received 8 January 2003; accepted 9 January 2004
KEYWORDS Glutamine; Protein turnover; Muscle; Stress; Glucocorticosteroids; Rats
Summary Introduction: Corticosteroid treatment affects muscle protein and glutamine metabolism. In the present study we aimed to clarify to what extent anorexia, weight loss and corticosteroids determine protein and glutamine metabolism in muscle. Methods: The study was performed in Wistar rats (300–350 g, n ¼ 40) given triamcinolone (0.25 mg/kg/day i.m.) treatment (CS group) for 14 days, sham treated free fed (FF group), sham treated pair fed (PF group) and sham treated pair weight (PW group). In vivo protein and glutamine turnover were measured using L-[2,63H]phenylalanine and L-[3,4-3H]glurtamine as tracer in a three compartment model across the hindquarter. Results: Corticosteroid treatment decreased total body weight to a greater extent than can be explained by decreased food intake only, justifying the need for pair weight controls. Muscle weight loss was relatively greater in the corticosteroid treated rats than in the pair weight controls indicating specific corticosteroid induced changes in muscle protein metabolism. Pair weight rats increased muscle net protein breakdown rates from 573 nmol 100 g body weight1 min1 to 1573 nmol 100 g body weight1 min1 (Po0:05 vs FF). In the corticosteroid treated rats net protein breakdown rates increased to 2274 nmol 100 g body weight1 min1 (Po0:01 CS vs FF/PF) Net protein breakdown in corticosteroid treated rats was accompanied by increased glutamine efflux from the hindquarter (Po0:05; CS vs FF/PF/PW). The latter could predominantly be explained by de novo synthesis. Furthermore, corticosteroid treatment induced a loss of plasma to free muscle glutamine gradient indicating down regulation of glutamine membrane transport rates into muscle. This effect was, however, similar in the pair weight control group and can thus be fully accounted for by the muscle weight loss. Conclusion: Two weeks treatment with triamcinolone increases net in vivo protein breakdown of muscle directly and indirectly due to secondary weight loss and decreased food intake. The amino acid residues are used for glutamine de novo
*Corresponding author. Department of Pediatric Surgery, UMC St. Radboud Hospital, PO box 9101, HP 413, 6500 HB Nijmegen, The Netherlands. Tel.: þ 31-24-3619761; fax: þ 31-24-3613547. E-mail address: [email protected] (I. de Blaauw). S0261-5614/$ - see front matter & 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.clnu.2004.01.004
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synthesis which is exported from muscle to visceral organs by down regulation of glutamine transport systems. These changes were in majority related to muscle weight loss. & 2004 Elsevier Ltd. All rights reserved.
Introduction Glucocorticoids have a prominent role in the catabolic response to sepsis, trauma and cancer. Chronic use of glucocorticoids in patients with inflammatory disease, e.g. Crohn’s disease, rheumatoid arthritis or chronic obstructive pulmonary disease, may also results in weight loss and muscle catabolism.1,2 In vitro, glucocorticoids induce muscle atrophy by increasing protein breakdown rates through activating the ATP-Ubiquitin-dependent pathways.3 In vivo, decreased protein synthesis rates have also been observed,4 but protein breakdown and synthesis have never been measured in vivo simultaneously. Furthermore, in previous experimental studies control groups were normally fed. Anorexia is one of the side effects of corticosteroid treatment and increased corticosteroid plasma levels.5 Anorexia can explain part of the muscle wasting. Therefore, pair fed controls should always be included. However, corticosteroids induce a greater weight loss than can be explained by anorexia only. Weight loss itself also induces changes in protein and amino acid metabolism of muscle. In vivo measurements of effects of chronic corticosteroid usage are thus measurements of direct corticosteroids effects but also indirect effects of weight loss induced by previous corticosteroid usage.6 Therefore, effects of corticosteroids should not only be compared with pair fed but also pair weight controls. Net protein breakdown of muscle induced by corticosteroids results in production of amino acids. These amino acids are taken up by visceral organs for gluconeogenesis and acute phase protein synthesis.7 The amino acid glutamine is consumed by visceral organs in the acute and chronic phase of inflammation not only for gluconeogenesis and protein synthesis but also for acid base homeostasis, glutathione synthesis and as energy substrate.7 The majority of glutamine is produced by hindquarter muscle. Recently, we described a model to measure in vivo glutamine consumption and production in muscle.8 Within this model it is also possible to determine in vivo de novo production. Our aim in the present study was to study in vivo changes in protein synthesis and breakdown rates of muscle induced by corticosteroids. We further aimed to study changes in in vivo muscle
glutamine metabolism induced by corticosteroids. The study was also conducted to clarify to what extent metabolic changes in muscle are caused by corticosteroids per se or by related food deprivation or weight loss.
Methods Animals Male Wistar rats (300–350 g, Harlan-Winkelmann, Borchen, Germany) were individually housed in metabolic cages during the experiment after an adaptation period of 3 days. Rats were given standard laboratory rat chow and subjected to standard 12 h light–dark cycle periods (7:30 A.M.– 7:30 P.M.) with room temperature maintained at 251C. The experiments were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals9 and approved by the Ethical Committee of Animal Research of the Maastricht University.
Experimental groups Rats (n ¼ 40) were randomly divided into four groups. 1. 2. 3. 4.
Free fed control group. Pair fed control group. Pair weight control group. Corticosteroid group.
The corticosteroid group received a daily injection of triamcinolone of 0.25 mg/kg body weight (1.25 mg/ml) intramuscular for 2 weeks. Triamcinolone is a fluorinated corticosteroid with increased anti-inflammatory potency. The dose given in the present study has previously been used as model to describe corticosteroid induced muscle atrophy and disturbances in energy metabolism.5 Food intake in the corticosteroid group was ad libitum. The free fed control group had food intake ad libitum. The pair fed group had equal food intake as the corticosteroid group. The pair weight group had comparable body weight as the corticosteroid group by adjusting daily food intake. All control groups received daily injections of 0.05 ml
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NaCl (150 mM) intramuscular for 2 weeks. All injections were given in the hamstrings of the left leg.
Study procedures Rats were randomly assigned to metabolic cages, taking an adaptation period of 4 days prior to the experiment into account. Hindquarter amino acid fluxes, protein and glutamine turnover were examined as described previously.8,10,11 In brief, postabsorptive, under ether anesthesia and at constant, pre-anesthesia body temperature, rats were subjected to a laparotomy. The right renal vein, the inferior caval vein and aorta were cannulated using a 25 gauge needle fixed in a Silastic tube (Silastic Medical Grade tubing 0.051 cm ID, 0.094 OD, Dow Corning Corporation, Midland, MO, USA) and fixed with cyano-acrylate. For flow measurements, the indicator dilution method with para-aminohippuric acid (PAH) was used (pH 7.4, iso-osmolaric, Sigma A 1422, St Louis, MI, USA). A primed constant infusion (0.75 ml 100 g bw1 h1, 5 mmol/l) of PAH was infused in the aorta, using a Minipuls 3 Peristaltic Pump (Gilson Medical Electronics Inc., Villiers-le-Bel, France). For protein and glutamine turnover studies, a primed constant infusion of L-[2,6-3H]phenylalanine and of L-[3,4-3H]Glutamine (NEN Dupont, NET-493, Mechelen, Belgium) was given in the right renal vein. 3H-Phenylalanine and 3 H-glutamine was given at a rate of 1 mCi 100 g bw1 h1. A minimum infusion time of 30 min was needed to reach steady state concentrations for PAH, 3H-phenylalanine and 3H-glutamine.11 The right carotid artery was cannulated during the equilibrium period with PE 50 catheter (Intramedic, 0.051 cm ID, 0.094 cm OD, Clay Adams, Parispany, NY, USA). The complete procedure took approximately 40 min. Blood (1.0 ml per catheter) was simultaneously sampled from the caval vein and carotid artery. All blood was collected in heparinized cups on ice. Hereafter, the right gastrocnemius muscle was dissected free, freezeclamped, put in liquid nitrogen and stored at 801C until further analysis. Gastrocnemius muscle from the left leg was prepared and weighted. For PAH determinations, 50 ml heparinized blood was added to 500 ml TCA 10%, vortexed and centrifuged at 8900g at 41C. The supernatant was frozen in liquid nitrogen and stored at 801C until further analysis. For hematocrit determinations a microhematocrit tube was filled with heparinized blood and centrifuged at 10,000g at room temperature. Hematocrit was read with a Micro Hematocrit
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Reader. Plasma was obtained by whole blood centrifugation at 8900g at 41C for 5 min. For ammonia determinations, 200 ml plasma was vortexed with 20 ml TCA 50%, put into liquid nitrogen and stored at 801C. To determine plasma amino acid specific activities and concentrations, 200 ml plasma was added to 8 mg 5-sulfosalicylic acid, vortexed, frozen in liquid nitrogen and stored at 801C. For the determination of tissue amino acid concentrations and specific activities, tissue was pulverized using a mortar and pestle precooled in liquid nitrogen. The tissue was further homogenized and deproteinized in a Mini-Beadbeater. Approximately 100 mg tissue was added to 400 ml SSA 5%, with 300 mg glass beads (diameter 1 mm), and beaten for 30 s. The homogenate was centrifuged at 41C at 11,000g and the supernatant frozen in liquid nitrogen and stored at 801C until further determinations. To determine tissue dry weight and water content approximately 200 mg pulverized tissue was freeze dried for 24 h in a Speedvac connected with a refrigerated condensation trap (Savant Instruments Inc., Farmingdale NY, USA).
Biochemical analysis Plasma ammonia and PAH were determined spectrophotometrically on a Cobas Mira S by standard enzymatic methods, using commercially available kits.10,11,8 Plasma and tissue amino acid concentrations and specific activity were determined by fully automated HPLC as described previously.12
Calculations Plasma flow across the hindquarter was calculated using PAH in the indicator dilution method.10,11,8 Substrate fluxes are calculated by multiplying the venous-arterial concentration differences with the mean plasma flow of the group and are expressed in nmol 100 g body weight1 min1. A positive flux indicates net release, a negative flux reflects a net uptake. Branched chain amino acids (BCAA) are the sum of valine, leucine and isoleucine. Total release of a-amino-nitrogen was calculated as the sum of the individual amino acids which are released by the hindquarter except taurine, arginine and alanine.12 Tissue concentrations of amino acids are expressed in mmol/l and derived by dividing the tissue homogenate amino acid concentration (mmol/kg wet weight) with the tissue water content. Whole body protein and glutamine turnover were determined by calculating the arterial dilution of tracer amino acid at steady state.13 Protein turnover rates were calculated by the A-V
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dilution of tritium labeled phenylalanine across the hindquarter calculated in a three compartment model.8,14,15 Intracellular disposal (D) and production (P) of phenylalanine can thus be calculated. Production of phenylalanine reflects protein breakdown and disposal reflects protein synthesis because muscle metabolism of phenylalanine is restricted to synthesis into and breakdown from protein.16 Intracellular production and disposal of glutamine were calculated using the same model.14,8 Knowing the relative amino acid concentration of phenylalanine and glutamine in muscle protein enables us to calculate glutamine production derived from protein breakdown and de novo synthesis.8,17
Statistical analysis Results are presented as mean7SEM. Statistical analysis was performed using the statistical package SPSS.18 Significance between groups was tested non-parametrically by Kruskal-Wallis test. Significance was considered present at Po0:05:
Results Food intake is depicted in Fig. 1. Corticosteroid treatment reduced food intake in the first five days.
Time (days) Figure 1 Food intake of different groups during the 2 week experiment; free fed (J), pair fed (n), pair weight (m) and corticoid treated rats (K).
Table 1
Food intake gradually returned to control values hence after. Body weight (Table 1, Fig. 2) decreased in the corticosteroid rats by 20% of the initial bodyweight. Bodyweight of the pair weight rats decreased similarly by 22%. Both control and pair fed rats had increased body weights. Gastrocnemius dry weights decreased both in the corticosteroid group and the pair weight group (Table 1). However, gastrocnemius weight expressed as percentage of body weight, was only significantly decreased in the corticosteroid group. Arterial ammonia decreased in both pair weight and corticosteroid treated rats (Table 2). Arterial phenylalanine only decreased in corticosteroid treated rats. Glutamine concentration remained unchanged. Total arterial amino acid concentration decreased in the pair weight and corticosteroid treated rats. Hindquarter plasma flow remained unchanged in all groups (Table 3). Ammonia efflux decreased in corticosteroid rats. Phenylalanine efflux increased in corticosteroid treated rats. Phenylalanine efflux increased to a similar extent in the pair weight rats indicating a similar net protein breakdown in both groups. Branched chain amino acid efflux from the hindquarter decreased in corticosteroid treated rats. Glutamine efflux increased in pair weight rats. In corticosteroid treated rats it increased significantly further (Po0:05 vs PW). Total a-amino nitrogen efflux increased in both pair fed and pair weight rats and increased most in corticosteroid treated rats. Intramuscular concentrations are depicted in Table 4. Intracellular ammonia decreased most in corticosteroid treated rats. Glutamine concentrations increased in the pair weight rats but not in the corticosteroid treated rats. Whole body protein turnover, given as Rate of Appearance, remained unchanged (Table 5). No significant changes were observed in hindquarter protein synthesis (disposal) and hindquarter protein breakdown. The difference (net balance), however, did increase in the control weight and corticosteroid treated rats. Whole body glutamine turnover remained unchanged in all groups (Table 6). Glutamine disposal by hindquarter muscle remained unchanged. Glutamine production
Body weight and gastrocnemius weight. FF
Total body weight Weight gastrocnemius Gastrocnemius/bodyweight
34377 1.8970.05 0.5670.01
PF 33473 1.8870.03 0.5670.01
PW
CS b
26674 1.5570.02b 0.5870.01
25175b,d,e 1.2670.05b,d,f 0.5070.01a,c,f
Values are mean7SEM. Body weight and gastrocnemius in grams. FF, free fed; PF, pair fed; PW, pair weight; CS, corticosteroid treated group. Kruskall-Wallis: a,bPo0:05; Po0:001 vs FF; c,dPo0:01; Po0:001 vs PF; e,fPo0:05; Po0:01 vs PW.
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by the hindquarter, however, increased in pair weight and corticosteroid treated rats. This was in majority an increase in de novo glutamine production. Glutamine net balance by hindquarter muscle increased in pair weight rats. In corticosteroid treated rats glutamine net balance increased significantly further (Po0:05 vs PW). Glutamine plasma-free muscle gradient is considered to be a function of membrane transport carriers.29,30 The plasma-free muscle gradient decreased to a similar extent in both pair weight and corticosteroid treated rats (Table 6).
Figure 2 Percentual weight change of different groups during the 2 week experiment; free fed (J), pair fed (n), pair weight (m) and corticoid treated rats (K).
Table 2
Arterial ammonia and amino acid concentrations.
Ammonia Phenylalanine BCAA Glutamine Glutamate Total a-amino nitrogen
FF
PF
PW
CS
34377 6973 404727 598720 8777 30027135
33473 6074 343729a 660742 7376 28227154
26674a 5872 358719a 652736 5672 2683769a
25175a 4372a 241715b,d,f 525720 108711 2007766b,d,f
Values are mean7SEM. Arterial concentrations in mmol/l. BCAA branched chain amino acids (valine, leucine, isoleucine). Kruskall-Wallis: a,bPo0:05; Po0:001 vs FF; c,dPo0:01; Po0:001 vs PF; e,fPo0:05; Po0:01 vs PW.
Table 3
Hindquarter blood flow, ammonia and amino acid fluxes.
Flow Ammonia Phenylalanine BCAA Glutamine Glutamate Total a-amino nitrogen
FF
PF
PW
CS
3.170.5 69720 574 72713 149798 1974 1547207
3.971.2 92727 1276 59721 1447128 2378 4847307
3.070.9 94729 1573 71711 3227130a 2073 4767215
4.070.8 16728a 2274b,d 13714a,h 5067140c,e,g 85718b,e,h 11277196c,e,g
Values are mean7SEM. Plasma flow in ml/100 g body weight/ min. Fluxes in nmol/100 g body weight/min. BCAA branched chain amino acids (valine, leucine, isoleucine). Kruskall-Wallis: a,b,cPo0:05; Po0:01; Po0:001 vs FF; d,e,fPo0:01; Po0:01; Po0:001 vs PF; g,hPo0:05; Po0:01 vs PW.
Table 4
Intramuscular ammonia and amino acid concentration.
Ammonia Phenylalanine BCAA Glutamine Glutamate Total a-amino nitrogen
FF
PF
PW
CS
1224779 6877 349718 2414751 685735 250227791
10427125 5273 317726 2517799 700770 248387361
876764b 5473 321721 3143799c 743769 255877570
397742c,f,i 4273b,e,g 251712b,d,g 24237190h 721763 226037556a,d,g
Values are mean7SEM. Intracellular concentrations are in mmol/kg wet weight. BCAA branched chain amino acids (valine, leucine, isoleucine). Kruskall-Wallis: a,b,cPo0:05; Po0:01; Po0:001 vs FF; d,e,fPo0:01; Po0:01; Po0:001 vs PF; g,h,iPo0:05; Po0:01; Po0:001 vs PW.
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Table 5 Whole body phenylalanine rate of appearance, hindquarter intracellular phenylalanine disposal, production and net balance.
Whole body rate Hindquarter Disposal Production Net Balance
FF
PF
PW
CS
595715
637744
606750
569722
2775 3375 574
2177 3375 1276
2175 3977 1573a
1575 3775 2274b,d
Values are mean7SEM. Data in nmol/100 gr body weight/min. Kruskall-Wallis: a,b,cPo0:05; Po0:01; Po0:001 vs FF; d,e,fPo0:01; Po0:01; Po0:001 vs PF;
g,h
Po0:05; Po0:01 vs PW.
Table 6 Whole body glutamine rate of appearance, hindquarter intracellular glutamine disposal, production, net balance and glutamine artery-muscle free ratio.
Whole body Rate Hindquarter Disposal Production De novo production Net balance Artery/free muscle ratio
FF
PF
PW
CS
51317426
55787442
60517471
55747336
246777 394761 329762 149798 0.2570.01
342782 4857118 4197110 1447128 0.2770.02
247734 5697112a 499796c 3227130a 0.2170.01a,c
302770 7297134a 6567100c 5067140c,e,g 0.2170.01a,c
Values are mean7SEM. Data in nmol/100 g body weight/min except glutamine artery/free muscle ratio. Kruskall-Wallis: a,b,cPo0:05; Po0:01; Po0:001 vs FF; d,e,fPo0:01; Po0:01; Po0:001 vs PF; g,hPo0:05; Po0:01 vs PW.
Discussion In the present study corticosteroid treatment increased weight loss to a higher extent than pair feeding. Pair weight controls are therefore the most appropriate controls. Corticosteroid treatment induced a relatively greater loss of muscle tissue than the muscle weight loss induced in pair weight controls. Steroids therefore did have specific catabolic effects on muscle. These include increased net protein breakdown of muscle and hindquarter glutamine efflux.
protein breakdown. Approximately 60% of this increase also occurred in the pair weight group. The muscle wasting after corticosteroid treatment is partly secondary to weight loss. Net protein breakdown of muscle resulted in a significant increased efflux of total amino acids by the hindquarter. In the corticosteroid group the increased efflux occurred in conjunction with decreased arterial total amino acid concentrations. This indicates that the clearance of amino acids from the arterial pool by visceral organs also increased.
Effects of corticosteroids on protein kinetics
Effects of corticosteroids on muscle glutamine kinetics
Corticosteroid induced muscle wasting has previously been associated with decreased protein synthesis rates.19 In other studies increased protein breakdown rates have been observed.3,20,21 All these studies, however, used free fed controls and measurements were mostly ex vivo. In our study protein synthesis and breakdown were simultaneously measured in vivo. We observed decreased protein synthesis and increased protein breakdown rates, although both not significantly. These subtle changes in protein synthesis and breakdown, however, did result in a significant increase in net
Approximately 30% of the amino acids released by hindquarter muscle is glutamine.22 This is confirmed in our study. Glutamine is considered a conditional essential amino acid in times of stress when plasma corticosteroid levels are elevated. Following corticosteroid treatment, interorgan glutamine flux is characterized by an increased glutamine efflux from muscle.23,24 We also observed an increased efflux after corticosteroid treatment in conjunction with increase in vivo glutamine production in muscle. In part glutamine production was derived from protein breakdown
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but the majority came from de novo synthesis. This is indeed consistent with increased glutamine synthetase enzyme activity and mRNA expression.25–27 De novo glutamine synthesis also increased in the pair weight group. The increase is thus not only a direct effect of corticosteroids but can in part be explained secondary to the weight loss. Fasting and muscle weight loss is known to increases glutamine synthetase activity.28 Plasma-tissue glutamine gradient was decreased similarly in the pair weight group and the corticosteroid group. Maintenance of plasma tissue gradient is a function of glutamine membrane transport carriers. These carriers are suggested to be under direct influence of corticosteroids.29,30 Previous studies showed that corticosteroids down regulate System Nn inward directed membrane transport24 and thus increase glutamine efflux from muscle with a decreased plasma-tissue gradient. In our study similar effects on plasma-tissue glutamine gradient were seen in the pair weight control and corticosteroid treated rats and the effect of corticosteroids could thus be fully explained by the weight loss.
Effects of steroids on other amino acids Glutamate is one of the precursors for glutamine de novo synthesis in muscle. The observed increase in glutamate uptake by muscle can only explain a small part of the increased de novo synthesis. Branched chain amino acids can also be converted to glutamine by muscle. However, branched chain amino acid uptake decreased in the corticosteroid treated rats. Therefore, the major part of branched chain amino acids used for glutamine de novo synthesis had to be derived from protein breakdown. BCAA contribute to approximately 20% of the amino acid residues after protein breakdown.24 Decreased intracellular BCAA concentrations were observed despite increased protein breakdown. This indicates that the BCAA are converted to other amino acids, most probably glutamine. In previous studies increased BCAA dehydrogenase activity has been observed in response to corticosteroids.31 The majority of glutamine production by muscle can thus be explained by de novo production from BCAA derived from protein breakdown.
Conclusion Two weeks treatment with the synthetic corticosteroid triamcinolone increases in vivo net protein
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breakdown of muscle. The amino acid residues are in majority used for glutamine de novo synthesis. Down regulation of glutamine membrane transport rates into muscle cells resulted in an increased glutamine efflux of hindquarter muscle. This was mostly a secondary effect due to weight loss. Glutamine clearance by visceral organs also appeared to be up regulated in response to corticosteroids.
Acknowledgements The authors wish to thank Mr. HMH van Eijk and Mr. DR Rooyakkers for analytical help and Mrs GAM ten Have for biotechnical assistance.
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