1 Department of Endocrinology, Growth hormone (GH) treatment of GH-deficient
adults increases lean body mass. To investigate this anabolic effect of
GH, body composition and postabsorptive and postprandial protein
metabolism were measured in 12 GH-deficient adults randomized to
placebo or GH treatment. Protein metabolism was measured after an
infusion of
[1-13C]leucine before
and after a standard meal at 0 and 2 mo. After 2 mo, there was an
increase in lean body mass in the GH group (P < 0.05) but no change in the
placebo group. In the postabsorptive state, there was increased
nonoxidative leucine disappearance (NOLD; a measure of protein
synthesis) and leucine metabolic clearance rate and decreased leucine
oxidation in the GH group (P < 0.05) but no change in the placebo group. After the meal, there was an
increase in NOLD and oxidation in all studies
(P < 0.05), but the increase in
NOLD, measured as area under the curve, was greater in the GH group
(P < 0.05). This study clearly
demonstrates for the first time that the increase in protein synthesis
in the postabsorptive state after GH treatment of GH-deficient adults
is maintained in the postprandial state.
protein synthesis; stable isotopes; feeding
GROWTH HORMONE (GH) is the classic anabolic hormone,
causing longitudinal bone growth in children (28). As GH secretion continues throughout life, there is considerable interest in the role
of GH in the regulation of body composition and metabolism in the
adult.
Compared with control subjects, adult GH-deficient patients have a
reduced lean body mass (LBM) of 7-8%, corresponding to ~4 kg of
lean tissue (24). Measurement of the cross-sectional area of the
dominant quadriceps muscle has demonstrated that in GH-deficient
patients, this muscle has a 15.5% smaller area than controls matched
for age, sex, and physical activity (9). In all reported studies of GH
replacement in adult GH-deficient patients, there has been an increase
in LBM after 6 mo despite the use of a range of different measurement
techniques (1, 2, 24). Studies that have investigated the effect of 3 yr of GH treatment suggest that the restoration of LBM is maintained
over longer periods (13). Because the maintenance of LBM in a range of
pathological conditions is associated with a favorable outcome, there
is intense interest in the anabolic action of GH.
Because LBM is predominantly protein, studies of protein metabolism
have been undertaken, using isotopic tracers, to elucidate the
mechanism of the anabolic action of GH. The majority of these studies
have been performed in the postabsorptive state, which is characterized
by a net loss of protein. However, most protein gain occurs in the
postprandial state, when circulating amino acid substrate levels are
increased and, during such conditions, repair and replacement of body
protein are achieved (17). In the postabsorptive state, the anabolic
effect of GH in adult GH-deficient patients has been shown, using
isotopic techniques, to be due to an increase in whole body protein
synthesis, with no significant effect on the rate of proteolysis (23).
However, the role of GH in the regulation of postprandial protein
metabolism is poorly understood, and there have been no previous
studies investigating this by the use of isotopic tracer techniques. We
have investigated the effect of GH treatment on postprandial protein
metabolism by using an infusion of
[1-13C]leucine in
GH-deficient adults in a double-blind placebo-controlled trial.
The study was a randomized double-blind placebo-controlled trial of
recombinant human GH (rhGH; Genotropin, Kabi Pharmacia, Stockholm, Sweden) in 12 patients with severe adult onset GH
deficiency. Postprandial whole body protein turnover was studied at 0 and 2 mo. Patient details are shown in Tables
1 and 2. All
patients had been GH deficient for 5 yr or more. The patients were a
subset of a larger group of 18 patients in whom postabsorptive
measurements of protein turnover and lipid concentrations were made on
the morning of the postprandial study. Those results have been reported previously (22, 23). GH deficiency was defined as a peak GH concentration lower than 3 ng/ml after the administration of a dose of
insulin that reduced the blood glucose to 2.0 mmol/l or less. Subjects
gave informed written consent to take part in the study, which had
received approval by the Ethics Committee of West Lambeth Health
Authority. All the patients were receiving appropriate adrenal,
thyroid, and gonadal hormone replacement therapy. There were no changes
made to these therapies during the study. The patients were in good
health with normal renal and hepatic function.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Table 1.
Patient characteristics
Table 2.
Patient details and measurements of glucose, insulin, C-peptide, and
NEFA
The dose of GH was 0.018 U/kg body wt daily (0.006 mg · kg1 · day
1)
for the first month, followed by 0.036 U · kg
1 · day
1
(0.012 mg · kg
1 · day
1)
for the second month. GH was self administered as a nocturnal subcutaneous injection. The placebo vials contained the same vehicle as
the rhGH vials and were indistinguishable from them. The target dose of
GH was 0.036 U · kg
1 · day
1,
but to avoid adverse effects, this dose was achieved over 1 mo.
Experimental protocol.
Subjects were studied on two consecutive days before and at the end of
2 mo of placebo or GH treatment. On both days, subjects fasted
overnight and took their usual hormone replacement therapy on the
morning of the study. Subjects requiring glucocorticoid replacement
therapy took 12.5 mg of cortisone acetate 2 h before the start of the
study. On day 1, patients received the
meal at 1200 without an isotope infusion to determine the effect of the meal on the isotopic enrichment of expired
CO2. Breath samples were taken
before and at intervals for 240 min after the meal. On
day 2, the study was performed between
0900 and 1600. While subjects were under local anesthesia, a cannula
(Venflon, Vigo, Helsingborg, Sweden) was placed in an antecubital vein
for administration of the infusates, and blood and breath samples were
taken to measure basal enrichment of plasma -ketoisocaproate
(
-KIC) and expired CO2. A
primed constant infusion (1 mg/kg; 1 mg · kg
1 · h
1)
of
L-[1-13C]leucine
was maintained for the duration of the study (
180 to 240 min). A
bolus dose of
NaH13CO3
(2.35 µmol/kg; 0.2 mg/kg) was given intravenously to prime the
bicarbonate pool. After a 150-min equilibration period to reach
steady-state tracer enrichment, the basal steady state was sampled
(
30 to 0 min) from a heated peripheral hand vein, and breath
samples were collected. The meal was given at 0 min. Further blood and
breath samples were taken at 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240 min. The mixed meal was designed to be as identical between
subjects and visits as possible and consisted of a cheese sandwich and
75 ml of Ensure Plus (Abbott Laboratories, Maidenhead, UK). The cheese
sandwich contained 40 g of cheese (St. Ivel, London, UK), 12 g of
butter, and two slices of whole meal bread (Harvest, London,
UK). All ingredients were from the same batch to ensure
uniformity. This meal provided 381 kcal, composed of 16 g of protein,
25 g of carbohydrate, and 25 g of fat. Height was measured with a
Harpenden stadiometer, and weight was recorded with subjects wearing
indoor clothing without shoes. Body composition was measured using the
technique of bioelectrical impedance (Holtain, Crymych, Wales, UK)
(14). This method has been shown to correlate well with estimates of
LBM by whole body counting of the 40K isotope of
potassium in GH-deficient adults (r = 0.91, P < 0.001) and GH-treated
GH-deficient adults (r = 0.93, P < 0.001) (20).
Analytic methods. The plasma amino acid profile was measured using an Alpha Plus II automated amino acid analyzer (Pharmacia, Cambridge, UK). Plasma immunoreactive insulin and C-peptide were measured by double-antibody radioimmunoassay (26) [within-assay coefficient of variation (CV) of 6 and 9%, respectively]. The C-peptide assay used an anti-C-peptide antiserum (GP-644) kindly donated by Professor A. Rubenstein (Dept. of Medicine, Univ. of Chicago). Plasma total insulin-like growth factor I was measured by radioimmunoassay after an ethanol-hydrochloric acid extraction (29) (within-assay CV of 7%). Plasma glucose concentration was measured on a Clandon Scientific glucose analyzer (within-assay CV of 2%) (Yellow Springs Instruments, Yellow Springs, OH). Nonesterified fatty acids (NEFA) were measured using a Cobas Mira Plus centrifugal analyzer (Roche) and a NEFA C ACS-ACOD kit (Wako Chemicals). The interassay CVs at 0.23 and 0.75 mmol/l were 7.5 and 3.6%, respectively. The intra-assay CVs at 0.23 and 0.75 mmol/l were 2.6 and 1.1%, respectively. Serum triglyceride concentrations were assayed enzymatically with a commercial test kit (Boehringer Mannheim). The between-assay CVs were 3 and 2.8% at 1.8 and 3 mmol/l, respectively.
PlasmaCalculations.
Leucine Ra and total leucine
disposal rate (Rd) were
calculated with the use of the one-compartment model originally
proposed by Steele (27), which has been validated for the measurement of leucine metabolism (32) and modified for stable isotopes. In this
model, -KIC was used as a measure of intracellular leucine enrichment (15). The tracer-to-tracee ratio for
-KIC was estimated with the use of the formulas of Cobelli et al. (8)
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Statistics. Results are expressed as means ± SE. AUC was measured by the trapezoid method. Statistical analysis was by ANOVA and paired t-test. P < 0.05 was taken as being significant.
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RESULTS |
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Measurements of the plasma concentrations of metabolites and hormones and leucine metabolism were not different in the two groups at 0 mo (Table 2). After 2 mo of GH treatment, there was a significant increase in body weight (P < 0.05) due to an increase in LBM (P < 0.05; Table 2). There was an increase in fasting insulin-like growth factor I (P < 0.005; Table 2) to the upper end of the aged-matched normal range (11.0-43.9 nmol/l, subjects aged 41-60 yr). There was an increase in C-peptide (P < 0.05) and NEFA (P < 0.05) (Table 2).
The measured CO2 tracer-to-tracee ratio in the baseline study for all patients on day 1 and day 2 is shown in Fig. 1. On day 1, there was a significant increase in CO2 tracer-to-tracee ratio after 90 min compared with the baseline value. Also shown in Fig. 1 is the rate of leucine oxidation calculated with and without the correction for the effect of the meal on CO2 tracer-to-tracee ratio. Failure to correct for the meal introduces a significant error of 7% (P < 0.01) in the calculation of leucine oxidation rate (calculated as AUCs). Leucine oxidation was therefore calculated in all patients before and after treatment with a correction for the meal effect.
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GH treatment increased basal NOLD and MCR (P < 0.03 and P < 0.05, respectively) and decreased basal leucine oxidation (P < 0.05), with a trend toward an increase in endogenous leucine Ra (P = 0.051) (Table 3). There was no significant change in basal leucine concentration. There were no significant changes in these measurements after 2 mo in the placebo group.
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After the meal, there was an increase in the plasma concentrations of glucose, insulin, and C-peptide (P < 0.001). Plasma triglyceride concentration was significantly increased (P < 0.05) at 240 min compared with baseline in both groups at 0 and 2 mo (placebo after 0 mo, 1.75 ± 0.24 vs. 2.64 ± 0.61 mmol/l; placebo after 2 mo, 1.65 ± 0.27 vs. 2.32 ± 0.41 mmol/l; GH after 0 mo, 1.88 ± 0.47 vs. 3.27 ± 0.78 mmol/l; GH after 2 mo, 1.74 ± 0.37 vs. 2.52 ± 0.72 mmol/l, respectively). There was no difference between the two groups in postprandial glucose, insulin, C-peptide, or triglyceride concentrations. There was a decrease in NEFA concentrations after the meal in both groups at 0 and 2 mo (P < 0.001, ANOVA). Despite the higher postabsorptive level of NEFA after 2 mo of GH, NEFA decreased to similar levels after the meal (240 min) in all studies (placebo after 0 mo, 0.16 ± 0.02 mmol/l; placebo after 2 mo, 0.16 ± 0.03 mmol/l; GH after 0 mo, 0.23 ± 0.03 mmol/l; GH after 2 mo, 0.24 ± 0.04 mmol/l).
After the meal there was an increase in leucine concentration, NOLD (Fig. 2), total leucine Ra (Fig. 3), and leucine oxidation (Table 3) in both groups (P < 0.05). The increase in NOLD, measured as AUC and total leucine Ra (exogenous+endogenous) AUC, was greater after GH treatment (P < 0.05; Table 3). Postprandial leucine MCR AUC was also greater in the GH-treated group (P < 0.03). In the GH-treated group, when corrected for the baseline values, the above-basal leucine Ra AUC for 240 min before (134 ± 142 µmol/kg LBM) and after GH treatment (161 ± 112 µmol/kg LBM) and NOLD AUC before (88 ± 92 µmol/kg LBM) and after GH treatment (107 ± 79 µmol/kg LBM) were not different. GH had no significant effect on postprandial leucine oxidation or leucine concentration (Table 3).
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DISCUSSION |
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This study clearly demonstrates for the first time that the increase in protein synthesis in the postabsorptive state after GH treatment of GH-deficient adults is maintained in the postprandial state. The significant increase in NOLD (a measure of whole body protein synthesis) and decrease in leucine oxidation in the fasted state in the GH-treated group confirm the previous reports of increased protein synthesis after GH treatment of GH-deficient adults in the postabsorptive state (3, 23, 25). After the meal, in both the GH-treated and placebo groups, there was an increase in leucine oxidation rate and an increase in NOLD, but the postprandial increase in NOLD was greater in the GH-treated patients. When corrected for the basal value, postprandial NOLD was similar before and after GH treatment, demonstrating that the postabsorptive increase in NOLD is maintained during feeding rather than further increased. Although previous studies have suggested that the postabsorptive increase in NOLD was the mechanism for the increase in LBM in these patients, the present study suggests that the anabolic effect of GH on protein synthesis in the postprandial state would also contribute to the increase in LBM.
Total leucine Ra (a measure of leucine appearance from the meal and endogenous leucine production from protein breakdown) was increased in both groups after the meal, but the increase was greater in the GH-treated patients. This may be due to greater uptake of amino acids from the gut after GH treatment or a higher endogenous leucine Ra (from protein breakdown).
The acute effect of feeding on protein metabolism has been studied extensively but has produced conflicting results. There is general agreement that amino acid oxidation is increased as in the present study but little agreement on whether food intake changes protein synthesis or protein breakdown. However, the measurement of the acute effect of feeding on whole body protein metabolism, using isotopes, is problematic. It requires the use of non-steady-state equations, and if protein breakdown is to be measured accurately, the Ra of amino acids from the gut must be quantified. This requires the protein in the meal to be labeled with an amino acid tracer in addition to an intravenous infusion of amino acid tracer. To avoid non-steady-state calculations, some investigators have studied the effect of feeding small hourly meals and used steady-state equations with either no measurement of amino acid absorption (12, 16) or with the measurement of amino acid absorption by spiking the meal with an amino acid tracer (4). These studies demonstrated that after feeding, protein synthesis either did not change (16) or increased by only a small proportion (12), whereas there was a pronounced decrease in protein breakdown (4, 12, 16). Although the calculation of protein metabolism in the steady state may be simpler and may make fewer assumptions, continuous feeding is not physiological. In addition, the absorption profile of an amino acid tracer added to a meal cannot provide an accurate measurement of the absorption profile of amino acids in a complex meal. Recently Boire et al. (5) have produced a labeled protein by infusing the udder of a cow with [13C]leucine and collecting the labeled milk protein, which they have then used to study the effect of feeding a single meal (6). This study demonstrated that an oral protein-bound amino acid tracer appears much more slowly in plasma than a free amino acid tracer. When protein metabolism was calculated with the use of the free amino acid tracer, feeding resulted in an 88% decrease in protein breakdown, whereas with the use of the protein-bound amino acid tracer, feeding was shown to increase protein synthesis with no effect on protein breakdown (6).
In the present study, we were particularly interested in the effect of GH on protein synthesis rather than proteolysis; thus it was decided to give a single (physiological) meal as opposed to either amino acid mixtures or multiple small meals. Because we did not have access to a labeled protein as used by Boire et al. (6), the meal did not contain an isotopic tracer. Although an isotopic tracer is required if exogenous and endogenous Ra components of total Ra are to be calculated, the calculation of NOLD only requires an accurate total Ra measurement. It was, however, very important that each meal was as identical as possible, and this was achieved by using the same constituents at each visit. At baseline and at 2 mo, each study was performed twice on consecutive days with and without tracer infusions of [1-13C]leucine. This was done to make accurate allowance for the isotopic change in endogenous 13CO2 excretion in response to the meal. If this was not performed, an error of 7% in the calculation of leucine oxidation and protein synthesis would have resulted.
After a meal, there is an increase in glucose, triglycerides, and amino acids and, in addition, an increase in insulin, all of which will play a role in the metabolic response. The different responses of protein metabolism to a meal, however, can probably be explained in terms of the insulin response to the meal and the tissue amino acid levels resulting from the protein content. An infusion of amino acids that increased circulating levels has been shown to markedly stimulate protein synthesis and amino acid oxidation (30), whereas an infusion of insulin in both normal subjects (7) and diabetic patients (18, 31) has been shown to decrease amino acid oxidation and inhibit protein breakdown with a resultant fall in amino acid levels. However, when this study was repeated in the presence of an amino acid infusion to prevent any fall (or rise) in amino acids during the insulin infusion, a similar inhibition of protein breakdown was demonstrated with no effect on protein synthesis (10, 21). Thus an increase in protein synthesis and oxidation after a meal may be an amino acid effect, and an inhibition of protein breakdown may be determined by the insulin response to the meal. The increase in leucine oxidation after a meal suggests that the amino acid effect to increase this must override the effect of insulin to decrease it (18, 31). In the present study, the meal, which consisted of a cheese sandwich and a drink of Ensure Plus (which contains protein in the form of casein and soya protein), resulted in a marked insulin response and a large rise in amino acid levels. In the GH-treated patients, the rise in amino acids was similar to the placebo group, suggesting that the greater increase in protein synthesis in this group was a direct effect of GH on protein synthesis rather than an effect on substrate supply. Thus it appears that GH may have an important role in the postprandial regulation of protein metabolism. The increase in basal leucine MCR, which is maintained in the postprandial state after GH treatment, suggests that GH may increase the efficiency of leucine removal from the circulation. Because GH has been shown to have a role in regulating amino acid transporters in the gut (13), it is possible that GH has a similar effect in peripheral tissues. This may be part of the mechanism by which GH increases protein synthesis.
In conclusion, this study demonstrates that GH stimulates protein synthesis in the postprandial state in addition to the postabsorptive state. Thus GH may have an important physiological role in the postprandial regulation of protein metabolism in adults.
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ACKNOWLEDGEMENTS |
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We thank Joanne Kelly, Elaine Albany, and Maggie Thomason for assistance with this study; Paul Forsey in the Hospital Pharmacy for preparing the isotopes; and Rolf Gunnarson and Kabi Pharmacia for supplying the GH.
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FOOTNOTES |
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This study was made possible by grants from the Special Trustees of St. Thomas' Hospital and the Hordern Fund.
Address for reprint requests: A. M. Umpleby, Dept. of Endocrinology, N Wing, 4th Floor, United Medical and Dental Schools, St. Thomas' Campus, London SE1 7EH, UK.
Received 16 June 1997; accepted in final form 6 February 1998.
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