- Email: [email protected]
Impact of glutamine infusions on muscle protein synthesis in fasted and endotoxin treated rats
Clinical Nutrition (1991) 10, SUPP: 43-46 0 Longman Group UK Ltd 1991
Impact of glutamine infusions on muscle protein synthesis in fasted and endotoxin treated rats M.M. JEPSON*
and D.J. MILLWARD
Nutrition Research Unit, DeDaftment of Clinical Science, London School of Hygiene and Tropical Medicine, 4 St Pancras Wai, London N WI ZPE, UK
Introduction
is a description of attempts to reproduce this effect in vivo using either glutamine itself, or the dipeptide alanyl-glutamine. The dipeptide can be used to overcome instability and insolubility which prevent inclusion of the amino-acid in commercial preparations for parenteral nutrition. Several highly soluble glutamine-containing dipeptides have now been synthesised, and shown to be rapidly hydrolysed in vivo, liberating free aminoacid for tissue metabolism (5).
The glutamine concentration of skeletal muscle is known to undergo marked changes in infection and other catabolic states; the remarkably high concentration of free glutamine in muscle (approximately 20mM in man) can show correspondingly large reductions of the order of 50%. To date, the explanation of the physiological significance of this mobilisation of muscle glutamine has centred on the role of glutamine as a labile pool of nitrogen which can be liberated in times of stress to provide carbon and nitrogen as substrate for the gut, rapidly proliferating lymphocytes and hepatic synthesis of acute-phase proteins (see Fig. 1). Until recently, less attention has been given to the physiological aetiology or consequences in muscle itself. Rennie and co-workers have now described a glutamine transporter in skeletal muscle (1) which is sensitive to a range of factors including ionic balance, hormones and nervous stimuli, all of which have been implicated in the mechanisms of altered protein turnover in catabolic states. These tindings, together with the observation that the fall in muscle glutamine concentration is correlated with the fall in protein synthesis in protein deficient and endotoxaemic rats (2, 3), raise the possibility that manipulation of the factors regulating the glutamine transporter and/or the muscle glutamine concentration [GLN], in times of stress, will allow control of the muscle protein balance. This paper is concerned with further analysis of the correlation between the reductions in muscle protein synthesis and [GLN] in rats (2, 3), in particular whether the relationship is coincidental or causal. McLennan et al (4) have demonstrated an increase in protein synthesis in the rat hind limb when it is perfused with glutamine. The following *Current address: Medical Research cent. London WIN 4AL. UK
Council.
20 Park
Methods The response to administration of alanylglutamine in endotoxaemic rats
Thirty male Sprague-Dawley rats weighing approximately 11Og were split into four groups as follows: 1) control; 2) endotoxin treated; 3) control plus alanyl-glutamine treatment for 40 or lOOmin; and 4) endotoxin plus alanyl-glutamine treatment for 40 or lOOmin. The endotoxin (E.coli lipopolysaccaride, 0127 B8, 3mg/kg) was injected 24h prior to death. Previous experiments which monitored the timecourse of changes in protein synthesis in response to this type and dose of endotoxin have shown that protein synthesis is maximally depressed at 24 h after a single injection (6). The alanyl-glutamine was dissolved in saline and adjusted to pH 6.5-7.0 with sodium hydroxide. A total dose of 6.2mMoVkg was administered as three separate intravenous (tail vein) injections, given at 15min intervals. Protein synthesis rate in the combined gastrocnemius and plantaris muscles was measured using the large dose phenylalanine method (6). The phenylalanine was injected 15min before the end of the 40 or lOOmin period over which the alanyl-glutamine was allowed to act. The results are shown in Figures 2A and 2B. The glutamine concentration in the control animals was 7.5 (SD0.7) mmoVkg wet weight, and this fell
Cres-
33
44
IMPACT OF GLUTAMINE
INFUSIONS ON MUSCLE PROTEIN SYNTHESIS
Biological role of muscle glutamine
marked fall in the muscle protein synthesis rate, indicative of a toxic response to excess ammonia * production (Fig. 3).
The response to administration of glutamine in fasted rats
Fig. 1. The biological role of muscle glutamine. Muscle glutamine is seen as a labile store of nitrogen and carbon for the gut, lymphocytes, kidney and liver in times of stress. The physiological consequences for muscle, including the control of protein turnover, have yet to be elucidated.
In order to move to a simpler model for depressed protein synthesis than the endotoxin-treated rat, the response of muscle protein synthesis to glutamine was examined in the fasted rat. In this experiment, 16 male Sprague Dawley rats weighing approximately 160g each were fasted for 24h. All rats were then anaesthetised (Hypnorm, 0.5 mg/kg) to facilitate constant infusion of gluta-
ALANYL-GLUTAMINE INFUSIONS 12
to 4.7 (0.9) mmol/kg in the endotoxin
treated rats (Fig. 2A). The administration of alanyl-glutamine to control rats resulted in significantly increased muscle [GLN] at both 40 and 1OOmin after the first injection (9.7 (1.6) mmoYkgat40minand9.9(1.1) mmol/kg at lOOmin). However, no such increases were observed in the endotoxin treated rats, in which [GLN] was 5.5 (1.0) mmol/kg at 40min and 5.7 (1.5) mmoYkg at lOOmin. In summary, injection of alanyl-glutamine did generate raised muscle [GLN] in control rats, but not in endotoxin treated rats. The fall in muscle [GLN] in endotoxin treated rats was associated with a fall in the rate of protein synthesis, as reported previously (2,3). Figure 2B shows the control rate of 14.7 (1.9) %/d, whilst the rate in endotoxaemic rats was only 9.1 (2.0) %/d. As might be anticipated, in view of the failure of the alanyl-glutamine injections to raise muscle [GLN], the rate of protein synthesis was not altered by the administration of dipeptide. Furthermore, the rise in [GLN] in the control rats was not associated with any increase in the rate of protein synthesis. If anything, the synthesis rate tended to be slightly reduced (13.5 (2.0) %/d at 40min, and 10.6 (0.9) %/d at lOOmin, versus 14.7 (1.9) %/d in controls). In a subsequent experiment, a 70% increase in muscle [GLN] was achieved in endotoxaemic rats via a much larger (17 mmol/kg) dose of alanylglutamine. However. this was associated with
muscle glutamine (mMolar)
!I,t 0
WMFVI.
6 6 4 3 2 1 0
40
0
100
time after treatment (mins)
protein synthesis 18r
0
rate (%/d)
40
rime after treatment Ml7a.lmlol.r/k# p.ptkbOn,50Ink,
100
(mins)
Fig. 2. The changes in muscle glutamine concentration (2A) and protein synthesis (2B) in control and endotoxin treated rats after injection of alanyl-glutamine.
CLINICAL NUTRITION
Response to ala-glu infusion muscle glutamine (mblolar) 12 ”
0
contmle
‘0
Ca
.ndotorln
n
.ndabJrk‘ (17 rnt*/klJ~
9 i YL
IUmMILpl I&2rYlh$d
ri
II
l-----l
lpaptld*
protein synthesis (%/d) l.s-
16-
12-
”
-p.ptm
pbptldm
Conclusions To summarise the results of both experiments, infusions of either glutamine or the dipeptide alanyl-glutamine were able to produce increased muscle [GLN] in control and fasted rats. However, in endotoxin treated rats a rise in [GLN] was much harder to attain, and required excessively high doses. Where an increase in [GLN] was achieved it was not associated with increased rates of muscle protein synthesis. The nature of the correlation between changes in muscle glutamine concentration and the rate of protein synthesis remains to be elucidated. Whilst our data appears to conflict with data using the perfused hind limb model, which shows a direct relationship between these two parameters (4), more work is needed before a conclusion can be reached concerning the nature of the relationship in vivo. Possible explanations for the inability to increase the rate of muscle protein synthesis in conjunction with the increase in glutamine include toxic effects of ammonia as a consequence of excessively high doses of glutamine, or alternatively, an effect of interference by the large amounts of phenylalanine which were used for the measurement of protein synthesis. Whilst the exact mechanism of this relationship has yet to be understood, the benefits in improved nitrogen balance following dipeptide administration to humans undergoing major surgery are becoming clear (7).
Fig. 3. Muscle glutamine concentration (3A) and protein synthesis (3B), 4Omin after a large dose (17mmolIkg) of alanyl-glutamine. The data shown for control and endotoxin treated animals which received the smaller dose of 6.2mMoVkg dipeptide is the same as that in Figure 2.
Giutamine protein synthesis 16-
14.
mine or saline via the tail vein. Six rats were infused with saline, and the remaining 10 were infused with glutamine (2mmol/kg) for 1 h. All rats were injected with a large dose of phenylalanine 15min prior to death for the measurement of protein synthesis in the combined gastrocnemius and plantaris (6). The results are shown in Figure 4. The infusion of glutamine caused a 32% increase in the muscle [GLN] compared with saline infused rats (5.4 (0.1) mmoYkg versus 4.1(0.5) mmol/kg). However, this was not associated with an increase in the rate of muscle protein synthesis (11.4 (1.6) %/d in glutamine infused versus 10.8 (1.3) %/d in controls).
CN
c
45
infusion
in fasted
rats
(9)/d)
1 + IdIne + gl”t*mln.
:
l2 _protein synthesis + ,o_ 7% increase (ns) *
,
* * * ++
*
8-
r 1
+ b
6-
muscle glutamine 4r
32% increase
2i oL_L_ 0
b_-__-. 1
2
__I_ 3
muscle
(p=O.O004)
4
~_.~ .I
5
6
~~ 7
i 8
glutamine (mmollkg)
Fig. 4. The response of muscle protein synthesis and glutamine concentration to a constant infusion of glutamine over 30min (total dose Zmmol/kg) in fasted rats.
46
IMPACT OF GLUTAMINE
INFUSIONS ON MUSCLE PROTEIN SYNTHESIS
Acknowledgements This work was supported by the Wellcome Trust and an ESPEN-AJINOMOTO Research Fellowship to M.M.J.
4.
5.
References 1. Rennie M J. Hundal H, Babij P et al 1986 Characteristics of a glutamine carrier in skeletal muscle have important consequences for nitrogen loss in injury, infection and chronic disease. Lancet ii: 10081012 2. Jepson M M, Bates P C. Broadbent P et al 1988 Relationship between glutamine concentration and protein synthesis in rat skeletal muscle. American Journal of Physiology 255: E166-172 3. Millward D J, Jepson M M and Omer A B 1989 Muscle glutamine concentration and protein turnover in vivo in
6.
7.
malnourished and endotoxaemic rats. Metabolism 38: l-8 MacLennan P A. Brown R A, Rennie M J 1987 A positive relationship between intracellular glutamine concentration and protein synthesis in rat skeletal muscle. FEBS letters 215: 187-191 Albers S, Wernerman J. Stehle P et al 1988 Availability of amino-acids supplied intravenously in healthy man as synthetic dipeptides. Kinetic evaluation of L-alanyl-Lglutamine and glycyl-L-tyrosine. Clinical Science 75: 463-468 Jepson M M, Pell J M, Bates P C, Millward D J 1986 The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats. Biochemical Journal 235: 329-336 Stehle P, Zander J. Mertes N et al 1989 Effect of parenteral glutamine dipeptide supplements on muscle glutamine loss and nitrogen balance after major surgery Lancet, Feb: 231-233
Impact of glutamine infusions on muscle protein synthesis in fasted and endotoxin treated rats M.M. JEPSON*
and D.J. MILLWARD
Nutrition Research Unit, DeDaftment of Clinical Science, London School of Hygiene and Tropical Medicine, 4 St Pancras Wai, London N WI ZPE, UK
Introduction
is a description of attempts to reproduce this effect in vivo using either glutamine itself, or the dipeptide alanyl-glutamine. The dipeptide can be used to overcome instability and insolubility which prevent inclusion of the amino-acid in commercial preparations for parenteral nutrition. Several highly soluble glutamine-containing dipeptides have now been synthesised, and shown to be rapidly hydrolysed in vivo, liberating free aminoacid for tissue metabolism (5).
The glutamine concentration of skeletal muscle is known to undergo marked changes in infection and other catabolic states; the remarkably high concentration of free glutamine in muscle (approximately 20mM in man) can show correspondingly large reductions of the order of 50%. To date, the explanation of the physiological significance of this mobilisation of muscle glutamine has centred on the role of glutamine as a labile pool of nitrogen which can be liberated in times of stress to provide carbon and nitrogen as substrate for the gut, rapidly proliferating lymphocytes and hepatic synthesis of acute-phase proteins (see Fig. 1). Until recently, less attention has been given to the physiological aetiology or consequences in muscle itself. Rennie and co-workers have now described a glutamine transporter in skeletal muscle (1) which is sensitive to a range of factors including ionic balance, hormones and nervous stimuli, all of which have been implicated in the mechanisms of altered protein turnover in catabolic states. These tindings, together with the observation that the fall in muscle glutamine concentration is correlated with the fall in protein synthesis in protein deficient and endotoxaemic rats (2, 3), raise the possibility that manipulation of the factors regulating the glutamine transporter and/or the muscle glutamine concentration [GLN], in times of stress, will allow control of the muscle protein balance. This paper is concerned with further analysis of the correlation between the reductions in muscle protein synthesis and [GLN] in rats (2, 3), in particular whether the relationship is coincidental or causal. McLennan et al (4) have demonstrated an increase in protein synthesis in the rat hind limb when it is perfused with glutamine. The following *Current address: Medical Research cent. London WIN 4AL. UK
Council.
20 Park
Methods The response to administration of alanylglutamine in endotoxaemic rats
Thirty male Sprague-Dawley rats weighing approximately 11Og were split into four groups as follows: 1) control; 2) endotoxin treated; 3) control plus alanyl-glutamine treatment for 40 or lOOmin; and 4) endotoxin plus alanyl-glutamine treatment for 40 or lOOmin. The endotoxin (E.coli lipopolysaccaride, 0127 B8, 3mg/kg) was injected 24h prior to death. Previous experiments which monitored the timecourse of changes in protein synthesis in response to this type and dose of endotoxin have shown that protein synthesis is maximally depressed at 24 h after a single injection (6). The alanyl-glutamine was dissolved in saline and adjusted to pH 6.5-7.0 with sodium hydroxide. A total dose of 6.2mMoVkg was administered as three separate intravenous (tail vein) injections, given at 15min intervals. Protein synthesis rate in the combined gastrocnemius and plantaris muscles was measured using the large dose phenylalanine method (6). The phenylalanine was injected 15min before the end of the 40 or lOOmin period over which the alanyl-glutamine was allowed to act. The results are shown in Figures 2A and 2B. The glutamine concentration in the control animals was 7.5 (SD0.7) mmoVkg wet weight, and this fell
Cres-
33
44
IMPACT OF GLUTAMINE
INFUSIONS ON MUSCLE PROTEIN SYNTHESIS
Biological role of muscle glutamine
marked fall in the muscle protein synthesis rate, indicative of a toxic response to excess ammonia * production (Fig. 3).
The response to administration of glutamine in fasted rats
Fig. 1. The biological role of muscle glutamine. Muscle glutamine is seen as a labile store of nitrogen and carbon for the gut, lymphocytes, kidney and liver in times of stress. The physiological consequences for muscle, including the control of protein turnover, have yet to be elucidated.
In order to move to a simpler model for depressed protein synthesis than the endotoxin-treated rat, the response of muscle protein synthesis to glutamine was examined in the fasted rat. In this experiment, 16 male Sprague Dawley rats weighing approximately 160g each were fasted for 24h. All rats were then anaesthetised (Hypnorm, 0.5 mg/kg) to facilitate constant infusion of gluta-
ALANYL-GLUTAMINE INFUSIONS 12
to 4.7 (0.9) mmol/kg in the endotoxin
treated rats (Fig. 2A). The administration of alanyl-glutamine to control rats resulted in significantly increased muscle [GLN] at both 40 and 1OOmin after the first injection (9.7 (1.6) mmoYkgat40minand9.9(1.1) mmol/kg at lOOmin). However, no such increases were observed in the endotoxin treated rats, in which [GLN] was 5.5 (1.0) mmol/kg at 40min and 5.7 (1.5) mmoYkg at lOOmin. In summary, injection of alanyl-glutamine did generate raised muscle [GLN] in control rats, but not in endotoxin treated rats. The fall in muscle [GLN] in endotoxin treated rats was associated with a fall in the rate of protein synthesis, as reported previously (2,3). Figure 2B shows the control rate of 14.7 (1.9) %/d, whilst the rate in endotoxaemic rats was only 9.1 (2.0) %/d. As might be anticipated, in view of the failure of the alanyl-glutamine injections to raise muscle [GLN], the rate of protein synthesis was not altered by the administration of dipeptide. Furthermore, the rise in [GLN] in the control rats was not associated with any increase in the rate of protein synthesis. If anything, the synthesis rate tended to be slightly reduced (13.5 (2.0) %/d at 40min, and 10.6 (0.9) %/d at lOOmin, versus 14.7 (1.9) %/d in controls). In a subsequent experiment, a 70% increase in muscle [GLN] was achieved in endotoxaemic rats via a much larger (17 mmol/kg) dose of alanylglutamine. However. this was associated with
muscle glutamine (mMolar)
!I,t 0
WMFVI.
6 6 4 3 2 1 0
40
0
100
time after treatment (mins)
protein synthesis 18r
0
rate (%/d)
40
rime after treatment Ml7a.lmlol.r/k# p.ptkbOn,50Ink,
100
(mins)
Fig. 2. The changes in muscle glutamine concentration (2A) and protein synthesis (2B) in control and endotoxin treated rats after injection of alanyl-glutamine.
CLINICAL NUTRITION
Response to ala-glu infusion muscle glutamine (mblolar) 12 ”
0
contmle
‘0
Ca
.ndotorln
n
.ndabJrk‘ (17 rnt*/klJ~
9 i YL
IUmMILpl I&2rYlh$d
ri
II
l-----l
lpaptld*
protein synthesis (%/d) l.s-
16-
12-
”
-p.ptm
pbptldm
Conclusions To summarise the results of both experiments, infusions of either glutamine or the dipeptide alanyl-glutamine were able to produce increased muscle [GLN] in control and fasted rats. However, in endotoxin treated rats a rise in [GLN] was much harder to attain, and required excessively high doses. Where an increase in [GLN] was achieved it was not associated with increased rates of muscle protein synthesis. The nature of the correlation between changes in muscle glutamine concentration and the rate of protein synthesis remains to be elucidated. Whilst our data appears to conflict with data using the perfused hind limb model, which shows a direct relationship between these two parameters (4), more work is needed before a conclusion can be reached concerning the nature of the relationship in vivo. Possible explanations for the inability to increase the rate of muscle protein synthesis in conjunction with the increase in glutamine include toxic effects of ammonia as a consequence of excessively high doses of glutamine, or alternatively, an effect of interference by the large amounts of phenylalanine which were used for the measurement of protein synthesis. Whilst the exact mechanism of this relationship has yet to be understood, the benefits in improved nitrogen balance following dipeptide administration to humans undergoing major surgery are becoming clear (7).
Fig. 3. Muscle glutamine concentration (3A) and protein synthesis (3B), 4Omin after a large dose (17mmolIkg) of alanyl-glutamine. The data shown for control and endotoxin treated animals which received the smaller dose of 6.2mMoVkg dipeptide is the same as that in Figure 2.
Giutamine protein synthesis 16-
14.
mine or saline via the tail vein. Six rats were infused with saline, and the remaining 10 were infused with glutamine (2mmol/kg) for 1 h. All rats were injected with a large dose of phenylalanine 15min prior to death for the measurement of protein synthesis in the combined gastrocnemius and plantaris (6). The results are shown in Figure 4. The infusion of glutamine caused a 32% increase in the muscle [GLN] compared with saline infused rats (5.4 (0.1) mmoYkg versus 4.1(0.5) mmol/kg). However, this was not associated with an increase in the rate of muscle protein synthesis (11.4 (1.6) %/d in glutamine infused versus 10.8 (1.3) %/d in controls).
CN
c
45
infusion
in fasted
rats
(9)/d)
1 + IdIne + gl”t*mln.
:
l2 _protein synthesis + ,o_ 7% increase (ns) *
,
* * * ++
*
8-
r 1
+ b
6-
muscle glutamine 4r
32% increase
2i oL_L_ 0
b_-__-. 1
2
__I_ 3
muscle
(p=O.O004)
4
~_.~ .I
5
6
~~ 7
i 8
glutamine (mmollkg)
Fig. 4. The response of muscle protein synthesis and glutamine concentration to a constant infusion of glutamine over 30min (total dose Zmmol/kg) in fasted rats.
46
IMPACT OF GLUTAMINE
INFUSIONS ON MUSCLE PROTEIN SYNTHESIS
Acknowledgements This work was supported by the Wellcome Trust and an ESPEN-AJINOMOTO Research Fellowship to M.M.J.
4.
5.
References 1. Rennie M J. Hundal H, Babij P et al 1986 Characteristics of a glutamine carrier in skeletal muscle have important consequences for nitrogen loss in injury, infection and chronic disease. Lancet ii: 10081012 2. Jepson M M, Bates P C. Broadbent P et al 1988 Relationship between glutamine concentration and protein synthesis in rat skeletal muscle. American Journal of Physiology 255: E166-172 3. Millward D J, Jepson M M and Omer A B 1989 Muscle glutamine concentration and protein turnover in vivo in
6.
7.
malnourished and endotoxaemic rats. Metabolism 38: l-8 MacLennan P A. Brown R A, Rennie M J 1987 A positive relationship between intracellular glutamine concentration and protein synthesis in rat skeletal muscle. FEBS letters 215: 187-191 Albers S, Wernerman J. Stehle P et al 1988 Availability of amino-acids supplied intravenously in healthy man as synthetic dipeptides. Kinetic evaluation of L-alanyl-Lglutamine and glycyl-L-tyrosine. Clinical Science 75: 463-468 Jepson M M, Pell J M, Bates P C, Millward D J 1986 The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats. Biochemical Journal 235: 329-336 Stehle P, Zander J. Mertes N et al 1989 Effect of parenteral glutamine dipeptide supplements on muscle glutamine loss and nitrogen balance after major surgery Lancet, Feb: 231-233