Effect of Leucine on Amino Acid and Glucose Metabolism in Humans. K. Sreekumaran Nair, Dwight E. Matthews, Stephen L. Welle, and Theodore Braiman.
Effect of Leucine K. Sreekumaran
on Amino
Acid and Glucose
Metabolism
in Humans
Nair, Dwight E. Matthews, Stephen L. Welle, and Theodore Braiman
Leucine has been reported to be an important regulator of protein metabolism. We investigated the effect of intravenous infusion of L-leucine versus saline on amino acid metabolism in eight healthy human subjects. Plasma concentrations of amino acids were measured and protein turnover was estimated using L-(1.r3C)lysine and L-(3,3,3-ZH3)leucine as tracers. Glucose kinetics were measured using o-(6,6-ZHz)glucose as a tracer. Leucine infusion increased the plasma leucine concentration from 103 f 8 to 377 f 35 prnol/L (P -z .Ol). Plasma concentrations of essential amino acids, including threonine, methionine, isoleucine, valine, tyrosine, and phenylalanine were significantly decreased by leucine infusion. Leucine infusion did not change lysine flux significantly (108 + 4 during saline Y 101 + 4 pmol! kg/ h-l during leucine infusion), but decreased lysine oxidation (13.2 + 0.8 v 10.7 f 1 pmol/kg/h, P c .05) and endogenous leucine flux (from 128 -t 4 to 113 2 7 pmol/kg/h, P < .05) when plasma (*Ha) ketoisocaproate (KIC) was used for calculation. During leucine infusion, the (*H3) KIC to (zH3) leucine plasma enrichment ratio increased from 0.76 f 0.02 to 0.88 -t 0.01 (P < .OOl), while estimation of leucine flux using plasma (sH3) leucine showed no change in endogenous leucine flux. Leucine infusion decreased hepatic glucose production and metabolic clearance of glucose, but did not change plasma concentrations of glucose, insulin, C-peptide, glucagon, epinephrine, norepinephrine, or free fatty acids. We conclude that leucine spares glucose and lysine catabolism and decreases plasma concentrations of essential amino acids. This study also demonstrated that the ratio of plasma KIC enrichment to leucine enrichment does not remain constant in all study conditions. Copyright 0 1992 by W.B. Saunders Company
L
EUCINE I-MS BEEN described as an in vivo regulator of protein metabolism in mammals.*-6 Sherwin’ demonstrated that L-leucine infusion decreased urinary nitrogen loss without altering urinary 3-methyl-histidine excretion, and proposed that leucine stimulates protein synthesis in humans. In vitro studies have shown that leucine increases protein synthesis’s*J and decreases protein degradation.1,2*6,9Other studies in rats failed to demonstrate any increase in protein synthesis by leucine,1° although administration of insuhn with leucine is reported to have enhanced protein synthesis.” In humans, we observed a significant correlation between plasma leucine concentration and protein synthesis estimated from the nonoxidative portion of leucine flux.‘* Contrary to these observations, it has been reported in humans that L-leucine meal increases net protein degradation across forearm muscle.13 Thus, the effect of leucine on protein turnover in humans remains to be clearly defined. In the current study, we used L-(l-W)lysine, an essential amino acid tracer, to follow changes in protein metabolism. This tracer should measure changes in protein breakdown without direct interference on the lysine pool from leucine infusion. Leucine kinetics were simultaneously determined using (*Hs)leucine and measurement of both plasma enrichment of leucine and cy-ketoisocaproate (KIC), with the latter representing the labeling of intracellular leucine. Finally, because leucine has been reported to affect glucose metabolism,7J4J5 and because glucose metabolism is intimately related to protein metabolism, we infused D-(6,6,2H2)glucose to measure glucose production.
MATERIALS
AND METHODS
Subjects We studied eight healthy human subjects (four men and four women) who ranged in age from 22 to 26 years (23.6 * 1 yr), with body weight ranging from 51.6 to 79.4 kg (65.3 t 4.6 kg) and height ranging from 164.9 to 181.5 cm (172.7 -t 2.5 cm). Their informed consent was obtained after explaining the protocol to them. The Metabolism, Vol41,
No 6 (June), 1992: pp 643-648
research was approved by the University of Rochester Research Subjects Review Board. Materials L-leucine (Ajinomoto USA, Raleigh, NC), 98 atom percent excess (APE) L-(1-13C)lysine, 98 APE r,-(3,3,3-*Hs)leucine, and 98 APE D-(6,6-*Hs)ghrcose (Cambridge Isotope Laboratories, Woburn, MA) were prepared using sterile precautions, passed through 0.22~pm filters (Millipore, Bedford, MA), and tested to be sterile and pyrogen-free before human use. Isotope purity of the solutions was tested by a gas chromatograph-mass spectrometer. Study Design Two separate studies were performed on each subject on different days. All subjects were admitted to the Clinical Research Center of the University of Rochester 48 hours before each study and placed on a weight-maintaining diet. Both studies were performed after an overnight fast. Infusions were performed in random order. Subjects received either a control infusion of 0.9% normal saline (NaCI) or 2% r.-leucine at a rate of 122 to 147 umol/kg/h (mean, 132 umolikglh) for 5.5 hours. During the first 30 minutes, the infusion rate was doubled to prime the leucine pool. One subject (no. 2 in Table 2) received 305 umol/kg/h of L-leucine. In four subjects, leucine was infused first, and in the other four, saline was infused first. Tracer infusions were started only after 1.5 hours of leucine or saline infusion, so that plasma concentrations of leucine would reach a steady state before beginning tracer infusions.
From the Division of Endocrinology, Metabolism, and Nutrition, Department of Medicine, College of Medicine, University of Vermont, Burlington, VT; the Department of Medicine and Surgery, Cornell University Medical College, New York, NY; and the Department of Medicine, University of Rochester, Rochester, NY. Supported by National Institutes of Health Grants No. DK-4197301, DK-38429, RR-000954, and RR-00044. Address reprint requests to K Sreekumaran Nair, MD, PhD, EndocrinelMetabolism Unit, Given C354, UVM ColIege of Medicine, Burlington, VT 05405. Copyright 0 1992 by W B. Saunders Company 00260495/92/4106-0011$03.00/0 643
NAIR
644
A plastic catheter was placed in a hand vein in a retrograde fashion for collection of arterialized venous samples. The hand was kept in a warm box (65” to 70°C) to arterialize venous blood. Another catheter was placed in the contralateral forearm vein for infusions. After baseline blood and expired-air samples were taken, bolus doses of L-(1-“C)lysine (3.5 kmol/kg), sodium (13C)bicarbonate (0.2 mgikg), L-(3,3,3-2H3)leucine (5.0 pmolikg), and D-(6,6-2H2)glucose (12.5 pmol/kg) were given to prime the respective pools and achieve an early plateau.1h-2n L-(1-13C)lysine (3.5 p,mol/kg/h), L-(3,3,3-*Hj)leucine (5.0 kmol/kg/h), and o-(6,6?H2)glucose (12.5 ~mollkgih) were then continuously infused for 4 hours. Blood samples were collected during the last 90 minutes at 270, 300, 315, 330, and 360 minutes for measurement of isotopic enrichments and substrate and hormone concentrations. Indirect calorimetry was performed during the last 90 minutes to measure carbon dioxide production. Sample Analysis and Calculations The isotopic abundances of plasma (“C)lysine, (‘H3)KIC, (“H3)leucine, and (2H2)g1ucose were determined using a gas chromatograph-mass spectrometer, as previously described.21 The isotopic abundance of 13COq _ was measured using an isotope ratio-mass spectrometer, as previously described.?’ Leucine flux was calculated from plasma (‘H,)leucine and (2H,)KIC abundances at plateau. Lysine flux and glucose flux were calculated from the plasma abundances of (I3 C)lysine and (2Hz)glucose, respectively, at plateau. The equation used for all of these steady-state flux calculations is as follows: Flux = i (EJ E, - 1) ~molikgih, where i is the infusion rate of tracer expressed as kmol/kg/h, E, is isotopic abundance (expressed as atom % excess) of the tracer, and E, is isotopic abundance (expressed as atom % excess) of tracer in plasma at plateau. Lysine oxidation was calculated using plasma (13C)lysine as the precursor pool. Lysine oxidation = F ‘“CO? (l/E, - l/E,) 100, where F 13C02 is the rate of production of 13COzfrom (13C)lysine. E, is plasma isotopic abundance, and E, is isotopic abundance of the tracer. In these calculations, it is assumed that the recovery of 13COzis 80%. Endogenous leucine flux was calculated by subtracting leucine infusion rate from leucine flux. Plasma concentrations of amino acids were analyzed by highperformance liquid chromatography (HPLC) with postcolumn ophthaldehyde derivatization. 22 Plasma concentrations of insulin (Insulin kit, Cambridge Nuclear, Los Angeles, CA), cortisol (Cortisol kit, Diagnostic Products, Los Angeles, CA), C-peptide,‘” and glucagon’4 were measured by radioimmunoassay. Epinephrine and norepinephrine were measured by radioenzymatic assay.25
more than the concentrations (P < .05) (Table 1).
of the other amino acids
Isotopic Enrichments An isotopic plateau during the last I hour is demonstrated in Fig 1. Slopes of different tracers were found to be not significantly different from zero (Fig 1). Leucine Flux
Endogenous leucine flux (estimated from plasma KIC isotopic abundance) during leucine infusion (112.8 i_ 7.5 bmol/kg/h) was lower (12.2% t 4.6%) than during saline infusion (129.5 2 4.0 pmol/kg/h, P < .05). This decrease in leucine flux was significant even after excluding subject no. 2, who received higher amounts of the L-leucine infusion. The ratio of (2H~)KIC enrichment to (2H3)leucine enrichment increased from 0.76 * 0.02 (saline infusion) to 0.88 ‘_’ 0.01 (leucine infusion) (P < .OOl). Endogenous leucine flux estimated from plasma (2H3 )leucine enrichment was not significantly affected by leucine infusion (Table 2). Lysine Kinetics
Changes in lysine flux during leucine infusion did not reach statistical significance (P < .l). There was a 20.7% + 4.9% reduction in lysine oxidation during leucine infusion in seven of the subjects (Fig 2). In one subject, there was an increase in lysine oxidation. The mean difference in lysine oxidation (13.2 A 0.9 kmolikglh during saline infusion and 10.7 t 1.0 pmol/kg/h during leucine infusion) was significant (P < .05) even after excluding subject no. 2, who received higher amounts of L-leucine. Nonoxidative disposal of lysine (lysine flux minus lysine oxidation) was not different between the two studies (95 5 5 pmol/kg/h during saline infusion v 93 2 3 kmol/kg/h during leucine infusion). The plasma concentration of lysine showed no significant difference between saline and leucine infusions. CO2 production during saline infusion (182 2 22 mL/min) was not different from that during leucine infusion (183 + 1 mL/min). Table 1. Effect of L-Leucine Infusion on Plasma Amino Acid Concentrations Treatment
Data Analysis Paired I tests were used to determine whether observed differences in mean values for all variables during the saline or L-leucine infusions were statistically significant. The measurements are expressed as the mean 4 SEM in the Results and in the tables. RESULTS Plasma Concentrations of Amino Acids
plasma concentrations
(~mol/L)
Amino Acid
Leucine infusion increased the plasma leucine from 103 * 8 to 377 * 35 kmol/L concentrations of threonine, methionine, tyrosine, and phenylalanine were lower infused than when saline was infused
concentration of (P < .Ol). Plasma isoleucine, valine, when leucine was (P < .05). The
of valine and isoleucine decreased
ET AL
Threonine
99 f 9
Serb
85 k 7
Glycine
143
Alanine
196 k 23
Methionine
16 5 2
lsoleucine
41 ? 3
Leucine
103 t
Valine
187
5
2 6
186 c 10 9 *
2”
12 k 2t 377
k 35*
78 t- 7t
40 + 3
18 5 3t
Phenylalanine
37 *
25 -c 2t
NOTE. i
8
k 16
76 i 135
Tyrosine
138
Lysine
*P
+ 12
75 2 4’
Results .05, tP
are mean < .Ol,
SP < .Ol, higher
lower
than
2
r 4
2 SEM. than
saline
saline
day.
day
134+5
LEUCINE AND PROTEIN TURNOVER
645
LEUCINE
5.5
SALINE
5.5
5.0
5.0
4.5
4.5
VI w
4.0
4.0
s
iii ::
3.5
3.5
8 E
* :
3.0
3.0
Y I
p
2.5
2.5
s “,
2.0
2.0
1.5
o-
i
1.0
1.5
--
1.0
s-
5 m
UI 8
n
::
::
ii5
Eiy
1 I
1
P
z
Fig 1. Isotopic enrichments of plasma (W)lysine, (*HI)KIC, (zH,)leucine, (*H,)glucose, and expired air WOz from 270 to 360 minutes. Calculations of flux and oxidation were performed using mean values from 300 to 360 minutes.
: 4
;
I
250
350
300 TIME
0
‘II,-Gluco#a
Glucose Metabolism
Hepatic glucose production showed a small but significant decrease during leucine infusion (P < .05), but plasma glucose concentration showed no change. The estimated metabolic clearance rate of glucose also decreased significantly during leucine infusion (P < .05) (Fig 3). Plasma Concentrations of Hormones
There was no significant change in the plasma concentrations of insulin, C-peptide, glucagon, epinephrine, norepinephrine, and cortisol during leucine infusion (Table 3). DISCUSSION
Infusion of L-leucine, in an amount approximately equivalent to endogenous leucine flux, decreased plasma concentrations of several essential amino acids (especially isoleucine and valine). It also decreased lysine oxidation without significantly altering lysine flux. Another effect of leucine was on glucose metabolism. Leucine decreased the
I
I
400250
(min) LX 0 C-LybIe
300
350 TIME
V
‘A,-KIC
b
4
400
(min)
‘Ha-Lauoin.
.
=co,
glucose production rate without altering plasma glucose concentration, thereby indicating a decrease of glucose utilization. These effects of leucine on glucose and lysine suggest sparing of other substrates by leucine. The cause of the profound effect of leucine on other essential amino acid concentrations is not entirely clear from our study or from other published data. One likely cause of the decrease in plasma concentrations of essential amino acids is a decrease in protein degradation. A decrease in protein degradation by exogenously infused leucine is consistent with in vitro studies.1-3,6 Infusion of leucine, isoleucine, and valine in a concentration lower than that used in the present study caused no change in leucine flux,r5 whereas infusion of an amino acid mixture has been reported to decrease leucine flu~i~,~~and to further reduce the insulin-induced leucine flux.16 Infusion of branchedchain amino acids was shown to decrease protein degradation in a recent study. 27Estimation of leucine flux based on plasma (2H3)KIC enrichment in the present study indicated
Table 2. Effect of L-Leucine Infusion on Leucine Flux in Eight Normal Subjects Plasma Leucine Concentration (wmol/Ll
Subject No.
Leucine Infusion
Endogenous Leucine Flux
Endogenous Leucine Flux
From (~HslLeucine
From (*H,)KIC
ipmollkglh)
Rate
(2HJKIC to 12H3)Leucine Ratio
(&mol/kglh)
Saline
Leucine
Saline
Leucine
1
153
381
124
109
116
145
138
0.76
0.83
2
106
615
305
97
132
134
106
0.78
0.89
3
109
418
133
94
101
122
74
0.79
0.87
4
89
339
135
99
97
124
118
0.81
0.90
5
73
293
147
92
99
120
116
0.80
0.92
6
86
297
122
108
100
118
100
0.67
0.89 0.87
(pmollkglh)
Saline
Leucine
Saline
Leucine
7
101
353
134
80
85
128
120
0.72
8
110
321
134
94
104
145
130
0.73
0.90
103.4 + 9
377.1 2 39.5t
154.3+-23.2
96.6 2 2.7
104.3 + 5.3
129.5 + 4
112.8k 7.5"
0.76 + 0.02
0.88 -tO.Olt
MeankSEM
lP < .05,lowerthanon salineday. tP
< ,001,
higherthansalineday.
NAIR ET AL
646
SALINE
LEUCINE
LEUCINE
SALINE
ah.1 f2.1
107.9 * 4.4
MEAN kSEM
101.5 i 4.2
18
.5 .O
MEAN iSEM
I I
I 2.24 io.19
MEAN fSEM
ai. f3.0
I 2.01* ztO.18
.
4.0
\ I
I
13.2 so.9
MEAN ztSEM
sE
3.5
2 : d
3.0
10.7* *l.O
2.5 2.0
.
.
Fig 2. Effect of leucine infusion on lysine flux and lysine oxidation. There is no significant change in lysine flux between saline day and leucine day. Lysine oxidation is lower on Ieuclne day than on saline day (P c X6).
a decrease in protein degradation. However, lysine flux, another indicator of protein degradation, was not significantly decreased by leucine. Alternatively, accelerated amino acid oxidation and increased protein synthesis could decrease plasma concentrations of essential amino acids. That there is no generalized increase in oxidation of other amino acids is demonstrated by a decrease in lysine oxidation in our study, and by the reported decrease in urinary nitrogen loss by 1eucine.l’ A lack of increase in nonoxidative disposal of lysine flux argues against an increase in protein synthesis. In another study, Schwenk and Haymondi5 reported a small ( < 2%) increase in nonoxidative leucine disposal by intravenous leucine infusion. In that study, the amount of leucine infused was less than half that of the current study and the isotope infusions were done differently, and therefore, the studies are not directly comparable.
1.5
1
2.76 iO.28
MEAN fSEM
I
2.47* kO.25
Fig 3. Effect of leucine infusion on glucose metabolism. There is no change in plasma glucose concentration, but glucose production rate and metabolic clearance rate of glucose are lower on leucine day than on saline day (p 4 .05).
Table 3. Effect of L-Leucine Infusion on Plasma Concentrations
of
Hormones Saline
Leucine
Insulin ($J/mL)
7.21 + 0.81
7.04 f 0.91 0.86 ? 0.16
Hormone
C-peptide (ng/mL)
0.82 -c 0.21
Glucagon (pg/mL)
83 k 3
79 2 2
Epinephrine (pg/mL)
93 2 23
82 r 22
Norepinephrine (pg/mL)
121 ‘t 20
116 k 16
Cortisol (rg/dL)
9.3 t 0.1
9.2 2 1.2
NOTE.
Results are the mean of three values obtained during the lest
30 minutes of the study (mean + SEM).
647
LEUCINE AND PROTEIN TURNOVER
Our study clearly demonstrates that the ratio of plasma leucine enrichment to KIC enrichment is not always constant, although a constant ratio has been observed in a variety of conditions.12*18,2B A change in this ratio, through intense exercise, has been previously reported in human subjects. It is clear from our study that infusion of unlabeled leucine alters the ratio of plasma enrichments of (13C)KIC to (r3C)leucine. Based on theoretical reasons and experimental evidence,28 it is likely that plasma KIC labeling represents that of intracellular leucine labeling. The intracellular leucine pool that is in isotopic equilibrium with KIC is a mixture of leucine, from intracellular protein degradation and leucine transportation into the cell from plasma. Exogenous infusion of a large amount of leucine will increase the amount of leucine transported across the cell membrane, and will narrow the difference between the isotopic enrichment of the intracellular and plasma compartments. It is therefore likely that plasma (t3C)leucine may represent intracellular (13C)leucine enrichment better when leucine is infused exogenously than it does without any exogenous leucine infusion. It is therefore incorrect to use plasma leucine enrichment to compare leucine flux estimated both with and without exogenous leucine infusion. Estimation of leucine flux based on plasma (13C)leucine suggests that unlabeled leucine entry into circulation is not changed by leucine infusion, whereas intracellular leucine appearance rate from protein breakdown was decreased. Increased leucine flux across the cell membrane (both ways) may explain this observation, although no definitive conclusions on this matter can be derived from the current study. The lysine tracer is independent of the pool changes occuring during leucine infusion. We therefore used the lysine tracer to give an independent measure of protein degradation. Lysine flux was not significantly decreased by leucine infusion, although there was a decrease in five of eight subjects. The lack of consistent change of lysine Aux during leucine infusion may be due to some inherent problems in using lysine as a tracer. Unlike (13C)leucine, (13C)lysine has no metabolite that reflects intracellular labeling. The lysine pool in the intracellular compartment is known to be large, and it takes a longer time for the label in the extracellular and intracellular compartments to reach an equilibrium. 29Therefore, plasma lysine enrichment may
not accurately represent the intracellular lysine enrichment, unless the tracer infusion is continued for a longer period than that used in this study. It is also likely that we underestimated the absolute value of lysine oxidation, since we do not have the intracellular lysine labeling to use as the precursor for oxidation. We observed a decrease in glucose production during leucine infusion, without changes in insulin and C-peptide levels. A lack of decrease in plasma glucose concentration while glucose production was decreased is consistent with a decrease in glucose disposal. Leucine in physiological concentration has been shown to inhibit the oxidation of D-(U-r4C)glucose in incubated skeletal muscle preparations.9 In addition, forearm studies showed a decreased glucose uptake during an intravenous infusion of leucine, isoleucine, and threonine.30 A decreased glucose utilization during leucine infusion has been indicated in another human study.31 A decrease in leucine flux and glucose production can occur as an effect of insulin, but we observed no effect of leucine on insulin or C-peptide levels. Moreover, the decrease in the glucose metabolic clearance rate and the lack of a decrease in plasma glucose are not consistent with an insulin effect. Leucine-induced insulin secretion has been reported only in studies in which a larger amount of leucine has been administered than that in our study.32-35 In summary, leucine infusion in normal subjects in the postabsorptive state decreased plasma concentrations of several essential amino acids, intracellular leucine flux (estimated from plasma KIC enrichment), lysine oxidation, glucose production rate, and metabolic clearance of glucose. These changes are not associated with any increase in insulin secretion. Based on the estimation of leucine flux from plasma (r3C)KIC, the leucine-induced decrease in plasma amino acids is due to a decrease in protein degradation. Further studies are needed to conclusively prove that leucine decreases protein degradation.
ACKNOWLEDGMENT
We are grateful to the nursing staff of the Clinical Research Center, to Mary Lorenson for analysis of plasma amino acids, and to Kirti Bhatt and David Robson for skilled technical assistance.
REFERENCES
1. Buse MG, Reid SS: Leucine: A possible regulator of protein turnover in muscle. J Clin Invest 56:1250-1261, 1975 2. Buse MG, Weigand DA: Studies concerning the specificity of the effect of leucine on the turnover of proteins in muscles of control and diabetic rats. Biochim Biophys Acta 475:81-89, 1977 3. Buse MG: In vivo effects of branched-chain amino acids on muscle protein synthesis in fasted rats. Horm Metab Res 13:502505,198l 4. Chang TW, Goldberg AL: Leucine inhibits oxidation of glucose and pyruvate in skeletal muscles during fasting. J Biol Chem 253:3696-3701,197s 5. Chua BL, Siehl DL, Morgan HE: A role for leucine in regulation of protein turnover in working rat hearts. Am J Physiol 239:E510-E514,1980
6. Poso AR, Wert JJ, Mortimore GE: Multifunctional control by amino acids of deprivation induced proteolysis in liver: Role of leucine. J Biol Chem 257:12114-12120,1982 7. Sherwin RS: Effect of starvation on the turnover and metabolic response to leucine. J Clin Invest 61:1471-1481.1978 8. Li JB, Jefferson LS: Influence of amino acid availability on protein turnover in perfused skeletal muscle. Biochim Biophys Acta 544:351-359, 1978 9. Fulks RM, Li JB, Goldberg AL: Effects of insulin, glucose and amino acids on protein turnover in rat diaphragm. J Biol Chem 250:290-298,1975 10. McNurlan MA, Fern EB, Garlick PJ: Failure of leucine to stimulate protein synthesis in vivo. Biochem J 204:831-838,1982
648
11. Garlick PJ. Grant I: Amino acid infusion increases the sensitivity of muscle protein synthesis in vivo to insulin. Biochem J 254579-584. 1988 12. Nair KS: Energy and protein metabolism in diabetes and obesity. Doctoral thesis, London, England, Council of National Academic Awards, 1984 13. Aoki TT, Brennan MF, Fitzpatrick GF, et al: Leucine meal increases glutamine and total nitrogen release from forearm muscle. J Clin Invest 68:1522-1528,198l 14. Devlin JT, Abumrad NN, Hoxworth B, et al: Extrapancreatic effects of L-leucine infusion in leucine-sensitive and control subjects. Metabolism 36:697-702,1987 15. Schwenk WF, Haymond MW: Effects of leucine, isoleucine. or threonine infusion on leucine metabolism in humans. Am J Physiol253:E428-E434.1987 16. Flak011PJ, Kulaylat M, Frexes-Steed M, et al: Amino acids augment insulin’s suppression of whole body proteolysis. Am J Physiol257:E839-E847, 1989 17. Sapir DG, Stewart PM, Walser M, et al: Effects of o-ketoisocaproate and of leucine on nitrogen metabolism in postoperative patients. Lancet 5:1010-1014, 1983 18. Matthews DE, Schwarz HP, Yang RD. et al: Relationship of plasma Ieucine and u-ketoisocaproate during a L-[1-r3C]leucine infusion in man: A method for measuring human intracellular leucine tracer enrichment. Metabolism 31:1105-1112.1982 19. Nair KS, Welle SL, Halliday D, et al: Effect of B-hydroxybutyrate on whole-body leucine kinetics and fractional mixed skeletal muscle protein synthesis in humans. J Clin Invest 82:198-205, 1988 20. Nair KS, Halliday D, Griggs RC: Leucine incorporation into mixed skeletal muscle protein in humans. Am J Physiol 254:E208E213,1988 21. Matthews DE, Motil KJ. Rohrbaugh DK. et al: Measurement of leucine metabolism in man from a primed, continuous infusion of L-[1-‘“Clleucine. Am J Physiol238:E473-E479, 1980 22. Hill DW, Walter FH, Wilson RD. et al: High performance liquid chromatographic determination of amino acids in the picomole range. Anal Chem 51:1338-1341, 1979
NAIR
ET AL
23. Heding LG: Radioimmunological determination of C-peptide in serum. Diabetologia 11:541-548, 1975 24. Nair KS, Halliday D, Matthews DE, et al: Hyperglucagonemia during insulin deficiency accelerates protein catabolism. Am J Physiol253:E208-E213,1987 25. Lilavivathana U, Brodows GR, Woolf PD, et al: Counterregulatory hormonal responses to rapid glucose lowering in diabetic man. Diabetes 28:873-877. 1979 26. Gelfand RA, Glickman MG, Castellino P, et al: Measurement of t_-[1-r4C]leucine kinetics in splanchnic and leg tissues in humans. Diabetes 37:1365-1372, 1988 27. Louard RJ. Barrett EJ. Gelfand RA: Effect of infused branched chain amino acids on muscle and whole body amino acid metabolism in man. Clin Sci 79:4_57-466,1990 28. Schwenk WF, Beaufrere B, Haymond MW: Use of reciprocal pool specific activities to model leucine metabolism in humans. Am J Physiol249:E646-E650, 1985 29. Waterlow JC. Garlick PJ. Millward DJ: Protein Turnover in Mammalian Tissues and in the Whole Body. Amsterdam, The Netherlands, North-Holland, 1978. pp 305-308 30. Schwenk WF, Haymond MW: Decreased uptake of glucose by human forearm during infusion of leucine, isoleucine, or threonine. Diabetes 36:199-204, 1987 31. Devlin JT, Brodsky I, Scrimgeour A, et al: Amino acid metabolism after intense exercise. Am J Physiol 258:E249-E255, 1990 32. Lundholm K, Edstrom S, Ekman L, et al: Protein degradation in human skeletal muscle tissue: The effect of insulin, leucine, amino acids, and ions. Clin Sci 60:319-326, 1981 33. Eriksson LS, Hagenfeldt L. Felig P, et al: Leucine uptake by splanchnic and leg tissues in man: Relative independence of insulin levels. Clin Sci 65:491-498.1983 34. Hagenfeldt L, Eriksson S, Waren J: Influence of leucine on arterial concentrations and regional exchange of amino acids in healthy subjects. Clin Sci 59:277-288,198O 35. Frexes-Steed M. Warner ML, Bulus N, et al: Role of branched-chain amino acids in regulating protein metabolism during fasting. Am J Physiol258:E907-E990. 1990
