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Medical Department M (Endocrinology and Diabetes) (H.N., N.M., J.S.C., J.O.L.J.), Aarhus Kommunehospital, Aarhus DK-8000, Denmark; and Endocrinology Division (K.S.N.), Mayo Clinic, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Helene Nørrelund, Medical Department M, Aarhus Kommunehospital, 8000 Aarhus C, Denmark. E-mail: helenenorrelund{at}dadlnet.dk
Abstract
The consequences of GH deficiency during conditions in which endogenous GH release is acutely stimulated are largely unknown. Short-term fasting constitutes a robust GH stimulus, but the metabolic significance of GH during fasting is uncertain.
To address both of these issues, we therefore evaluated the effect of GH on substrate metabolism during fasting in adults with GH deficiency. Seven hypopituitary GH-deficient patients were each studied twice during a 40-h fast: once with GH replacement continued and once with GH discontinued during the fast. After 40 h of fasting, protein synthesis and turnover were higher with than without GH replacement [phenylalanine incorporation (µmol/kg fat free mass/h): 36.6 ± 1.2 (GH) vs. 32.8 ± 1.4, P < 0.05; phenylalanine flux (µmol/kg fat free mass/h): 41.3 ± 1.0 (GH) vs. 38.0 ± 1.8, P < 0.05]. During continued GH replacement, urea excretion decreased during nighttime [urea excretion (mmol/24 h): 269 ± 51 (GH) vs. 390 ± 69, P < 0.05], and a significant decline in urea-N synthesis rate was found [urea-N synthesis rate (mmol/h): 14.7 ± 1.6 (GH) vs. 21.1 ± 2.2, P < 0.01]. GH replacement was associated with increased lipid oxidation [lipid oxidation (mg/kg per min): 0.91 ± 0.07 (GH) vs. 0.70 ± 0.03, P < 0.05]. Finally, continuation of GH induced moderate elevations in plasma glucose levels without significant changes in total glucose turnover or oxidation.
In summary, continued GH substitution during fasting conserves nitrogen, which involves stimulation or maintenance of protein synthesis. Our data support the importance of GH replacement in hypopituitary adults.
NUMEROUS STUDIES HAVE shown that GH replacement in hypopituitary adults with GH deficiency increases lean body mass and reduces fat mass (1, 2, 3, 4). Studies of such patients in the postabsorptive state suggest that GH stimulates protein synthesis without altering protein turnover (5). In healthy adults these effects of GH seem to act directly at the target tissue [i.e. skeletal muscle (6, 7)], but it is also plausible that stimulation of lipid oxidation indirectly contributes to the protein anabolic effects of GH in GH-deficient patients (8).
The fact that fasting constitutes a very robust stimulus for pituitary GH release (9) suggests a physiological role of GH during this condition, but experimental data to substantiate this hypothesis are surprisingly few. Short-term fasting in normal adults is characterized by increased lipolysis, but gluconeogenesis is also transiently elevated to meet glucose utilization by the brain. Hypoglycemia during fasting is a regular occurrence in untreated GH-deficient children (10), but whether this is associated with abnormalities in protein and lipid metabolism is unknown.
A study of substrate metabolism during short-term fasting in GH-deficient adults has so far not been conducted. Such a protocol would provide new information about the metabolic effects of GH in general and the consequences of GH deficiency in particular.
The present study was designed accordingly to assess the role of GH in the regulation of substrate metabolism during short-term fasting in GH-deficient adults. The patients underwent 40 h of fasting on two occasions with and without concomitant GH replacement, respectively. The methods included measurements of urea excretion, indirect calorimetry, and isotopic determination of the turnover of whole body glucose, phenylalanine, and tyrosine.
Subjects and Methods
Seven hypopituitary GH-deficient adults (one female and six
males) with a mean age of 45.6 ± 4.3 (SE) years and a
mean body mass index of 26.2 ± 1.6 kg/m2
participated. The patients had been on stable replacement therapy,
including GH, for at least 1 yr and were taking thyroid, adrenal, and
sex hormone replacement when appropriate. None of the patients had
acromegaly, Cushing’s disease, or diabetes. The diagnosis of GH
deficiency was ultimately based on two GH stimulation tests. The
clinical studies were performed in Aarhus, Denmark. All patients gave
informed consent to participate in the study, which had been approved
by the Ethical Committee of Aarhus County. The patients were each
studied twice during 40 h of fasting (i.e. overnight
plus 28 h) with conventional GH replacement
(Norditropin, Novo Nordisk, Copenhagen,
Denmark), injections after 1 and 25 h of fasting (2100 h) [dose,
1.3 ± 0.1 IU/day (
0.4 mg/day)] or discontinuation of the two
regular evening GH injections, respectively (Fig. 1
). The two studies were done in random
order. Blood was sampled at the following time points: 12, 13, 15, 19,
23, 27, 31, 35, 36, 37, 38, 391/2, 393/4, and 40 h
after the beginning of the fast. During fasting only tap water was
allowed.
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Substrate metabolism was investigated during the last 4 h.
After baseline blood sampling priming doses of
[3-3H]glucose (NEN Life Science Products, Boston, MA) (20 µCi),
L-[15N]-phenylalanine
(0.7 mg/kg),
L-[2H4]-tyrosine
(0.5 mg/kg) and
L-[15N]-tyrosine
(0.3 mg/kg) (Cambridge Isotope Laboratories, Inc.,
Andover, MA) were given. A continuous infusion of
[3-3H]-glucose (20 µCi/h),
L-[15N]-phenylalanine
(0.7 mg/kg per hour) and
L-[2H4]-tyrosine
(0.5 mg/kg per hour) was started and maintained for 4 h. The
chemical, isotopic, and optical purity of the isotopes was tested
before use. Solutions were prepared under sterile precautions and were
shown to be bacterial and pyrogen free. The specific activity of
tritiated glucose was assayed as previously described
(11).
L-[15N]-phenylalanine,
L-[2H4]-tyrosine,
and
L-[15N]-tyrosine
were measured as their t-butyldimethylsilyl ether derivates under
electron ionization conditions (12). Plasma concentrations
of amino acids were determined by an HPLC system (HP 1090 series 2
HPLC, 1046 fluorescence detector and cooling system;
Hewlett-Packard, Palo Alto, CA) with precolumn
O-phthalaldehyde derivatization (13). In
addition, concentrations of phenylalanine and tyrosine were measured by
mass spectrometry using ß-methylphenylalanine and 
-methyltyrosine,
respectively, as internal standards (12). Fat free mass
(FFM), comprising both muscle tissue and nonmuscle fat-free tissue, was
estimated by means of a dual-energy x-ray absorptiometry scanning
(QDR-1000/W, Hologic, Inc., Waltham, MA). Plasma glucose
was measured in duplicate immediately after sampling on a glucose
analyzer (Beckman Coulter, Inc., Palo Alto, CA). A double
monoclonal immunofluorometric assay (Delfia, Wallac, Inc.,
Turku, Finland) was used to measure serum GH. Plasma glucagon
(14), and serum insulin-like growth factor I (IGF-I)
levels (15) were measured by RIAs. Free fatty acids (FFA)
were determined by a colorimetric method employing a commercial kit
(Wako Chemicals, Neuss, Germany), while glycerol and
3-hydroxybutyrate were analyzed by autofluorometric enzymatic methods
(16). Catecholamines were measured by liquid
chromatography (17). Urea excretion was determined by an
indophenol method and serum urea by a commercial kit (COBASINTEGRA,
Roche, Hvidovre, Denmark). Insulin was determined by a
commercial enzyme-linked immunosorbent assay (DAKO Corp.,
Glostrup, Denmark), while cortisol was measured by an automated
chemiluminescence system (Chiron Corp., Fernwald,
Germany). Thyroid hormones were measured by radioimmunoassays as
previously described (18, 19). Indirect calorimetry
(Deltatrac monitor, Datex Instrumentarium, Helsinki,
Finland) was performed for 30 min (26
–27 h),
allowing measurements of energy expenditure and the respiratory
exchange ratio. The initial 5 min of calorimetry were used for
acclimatization, and calculations were based on mean values of 25 1-min
measurements. Net lipid and glucose oxidation rates were calculated
from the above measurements (20). Net nonoxidative glucose
disposal was calculated by subtracting oxidative glucose disposal
(glucose oxidation rates) from total glucose disposal measured
isotopically. Urine for determination of urea was collected in three
fractions: (1) 12–28 h (2) 28–36 h, and (3) 36–40 h (Fig. 1
). Urea
accumulation was determined assuming immediate dispersion of urea from
the blood to total body water (21). Urea-N synthesis rate
(UNSR) was determined as urinary excretion (28–36 h) of urea corrected
for accumulation of urea in total body water and for hydrolysis in gut
(22). UNSR was calculated as urinary excretion rate (E),
corrected for accumulation (A) in total water and for the fractional
intestinal loss (L): UNSR = (E+A)/(1-L), where E = (urine
flow, l/h) x (Urinary urea-N, mmol/L), A = (change in blood
urea-N, mmol/L per hour) x (total water, l). L was estimated to
be 0.14.
Data on hormones and metabolites during the tracer study are based on measurements within the last 30 min of the study.
Phenylalanine and tyrosine kinetics
For measurements of phenylalanine kinetics, the equations
described by Thompson et al. (23) were used.
Phenylalanine flux (Qp) and tyrosine flux were calculated as
follows:
 |
The rate of phenylalanine conversion to tyrosine
(Ipt) was calculated as follows:
 |
As a measure of protein synthesis, phenylalanine incorporation into protein is calculated by subtracting Ipt from Qp, because phenylalanine is irreversibly lost either by its degradative pathway via its conversion into tyrosine or by incorporation into protein.
Statistics
Results are expressed as the mean ± SEM.
Differences in the total area under the curves and data based on means
of duplicate/triplicate measurements within the last 30 min of the
study were analyzed by Student’s paired t test. Because GH
was injected at bedtime (2100 h) and serum GH peaked at 0300 h,
the effect of GH on lipids and urea excretion was also investigated
separately during nighttime (28–36 h of fasting) (Fig. 1). Data were
log transformed when not normally distributed as tested by
Kolmogorov-Smirnov. A P value below 0.05 was considered
significant.
Results
Circulating hormones (Table 1)
After 40 h of fasting, levels of GH [GH (µg/L): 0.56
± 0.16 (GH) vs. 0.23 ± 0.09, P <
0.05] and IGF-I [IGF-I (µg/L): 246 ± 39 (GH) vs.
165 ± 45, P < 0.05] were higher with than
without GH replacement. Circulating levels of insulin (Fig. 2), glucagon, norepinephrine, and
epinephrine were comparable; free T3 and total
T3 were increased during fasting with GH
replacement, whereas a tendency for a reduction in plasma cortisol was
observed (P = 0.08).
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Protein turnover (Fig. 3), expressed
as phenylalanine flux, was higher with GH substitution [phenylalanine
flux (µmol/kg FFM/h): 41.3 ± 1.0 (GH) vs. 38.0
± 1.8, P < 0.05], and the difference in tyrosine
flux almost reached significance [tyrosine (µmol/kg FFM/h):
30.0 ± 1.1 (GH) vs. 29.4 ± 1.3,
P = 0.07]. Phenylalanine incorporation into protein
was significantly higher during fasting with GH substitution
[phenylalanine incorporation (µmol/kg FFM/h): 36.6 ± 1.2 (GH)
vs. 32.8 ± 1.4, P < 0.05], whereas
phenylalanine degradation was comparable [phenylalanine hydroxylation
(µmol/kg FFM/h): 4.7 ± 0.6 (GH) vs. 5.2 ± 0.8,
P = 0.6]. Phenylalanine clearance rate was identical
in both situations [clearance rate (mL/min): 80 ± 11 (GH)
vs. 77 ± 16, P > 0.05].
|
. A significant reduction in the
concentration of several amino acids was found during GH
replacement.
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).
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Energy expenditure (Fig. 5),
measured by indirect calorimetry, was higher with GH replacement [EE
(kcal/24 h): 1750 ± 129 (GH) vs. 1581 ± 116,
P < 0.01]. Lipid oxidation was also higher during GH
replacement [lipid oxidation (mg/kg per min): 0.91 ± 0.07 (GH)
vs. 0.70 ± 0.03, P < 0.05] even
though the respiratory exchange ratio did not differ significantly
[respiratory exchange ratio: 0.795 ± 0.01 (GH) vs.
0.800 ± 0.002, P > 0.05 ]. Circulating lipid
fuel substrates tended to be higher in the GH replaced group because
FFA was increased 17% [FFA (mmol/L): 0.83 ± 0.07 (GH)
vs. 0.71 ± 0.06, P = 0.1] and plasma
glycerol was 40% higher [glycerol (µmol/L): 18.9 ± 2.1 (GH)
vs. 13.5 ± 1.2, P = 0.2] (Fig. 6). Plasma lactate was identical in both
situations [lactate (µmol/L): 752 ± 27 (GH) vs.
727 ± 32, P > 0.05]. Plasma glucose (Fig. 2)
was higher during GH replacement [glucose (mmol/L): 4.5 ± 0.2
(GH) vs. 4.1 ± 0.1, P < 0.01].
Glucose oxidation [mg/kg per min: 0.87 ± 0.14 (GH)
vs. 0.79 ± 0.02, P > 0.05] and
nonoxidative glucose turnover [mg/kg per min: 0.67 ± 0.14 (GH)
vs. 0.65 ± 0.11, P > 0.05] were
comparable in both studies (Fig. 5).
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Fasting is characterized by progressive fuel depletion and augmented secretion of GH. Surprisingly few controlled studies of the metabolic impact of GH during fasting have been conducted, and the present study was accordingly designed to assess the effects of GH on substrate metabolism with particular reference to the effect on protein metabolism. The phenylalanine turnover technique enabled whole-body rates of protein turnover, protein synthesis, and phenylalanine degradation to be estimated. The major finding was that discontinuation of GH replacement during fasting was associated with increased whole-body protein loss, which was accounted for by a net reduction in protein synthesis despite a moderate reduction in protein turnover.
Our study did not include a comparison between fasting and nonfasting, but the isolated effects of starvation on protein dynamics have previously been published in the literature. An increase in leucine flux (reflecting proteolysis) has been demonstrated after 30 h of fasting (24), and more prolonged fasting increases both leucine flux and oxidation (25) and prompts increased leucine release across the forearm (26). During short-term fasting branched-chain amino acid levels increase (consistent with increased proteolysis), whereas a decrease in other amino acids, reflecting reduced amino acid synthesis or increased utilization of amino acids for gluconeogenesis, has been reported (25, 27, 28). It is therefore plausible that the increased rate of protein synthesis observed during fasting with continued GH replacement still represents a decrease in protein synthesis relative to postabsorptive conditions.
We decided to study the effect of GH during ordinary treatment (i.e. bedtime injections), reasoning that this would be the habitual GH condition. It is noteworthy that the significant effects of GH on substrate metabolism were recorded approximately 9 h after the nightly GH peak.
A number of studies have been conducted to address the question whether exogenous GH conserves protein during caloric restriction. Studies in hypopituitary children (10) and obese adults (29, 30) have shown a clear nitrogen-retaining impact of GH as judged by urinary nitrogen loss, and GH has been reported to suppress hepatic ureagenesis specifically (31). Previous studies assessing the impact of GH on protein kinetics, which have been performed postabsorptively, have in general shown that GH primarily increases protein synthesis at whole-body level (5, 32, 33), and there is evidence that GH may increase muscle protein synthesis acutely (6, 7). On the other hand, some studies have failed to detect any effect of GH on muscle protein synthesis (34, 35). In the present study, whole-body turnover rates were measured representing an average of synthesis rates in different tissues. It is therefore possible that protein synthesis exhibited regional differences, which our method is unable to delineate (36). Furthermore, because excretion of urea was prominently reduced during the nightly GH peak, a measure of protein turnover at this time might have revealed more pronounced differences in isotopically assayed protein turnover.
A recent study of protein turnover in GH-deficient adults has demonstrated reduced rates of protein synthesis and breakdown and subsequent normal net protein loss, compared with normal controls (37), in line with earlier observations (38). An initial decline in lean body mass (LBM) may be the consequence of GH insufficiency, but clinical experience suggests that LBM stabilizes at a reduced level and this adaptation may explain the development of stable, albeit reduced, protein turnover and LBM in GH deficiency (37). GH substitution for 6 weeks in GH-deficient adults revealed increased net protein synthesis and unaltered total protein turnover (5). Our study in GH-deficient patients differed from those discussed above in terms of design and assay methodology, but it appears evident that GH preferentially stimulates protein synthesis both postabsorptively and during short-term fasting.
In the present study, a reduction in the plasma concentration of several amino acids was observed with continuation of GH replacement, which presumably reflects increased cellular uptake as protein synthesis is increased. Fryburg et al. (6) has demonstrated an increase in tissue uptake for amino acids during GH treatment in the absence of any significant change in release. With continued GH replacement, higher levels of lipid intermediates were demonstrated in our study. Fery et al. (39) has evaluated the role of fat-derived substrates in the regulation of gluconeogenesis during fasting. During fasting both FFA and ketone bodies tend to suppress gluconeogenesis and to protect the protein stores, and this is why decreased utilization of amino acids for gluconeogenesis can be expected.
The IGF-I level was significantly lower during fasting without GH replacement. The effects of IGF-I administration on whole-body protein metabolism seem to depend on ambient amino acid levels in the sense that IGF-I administered alone suppresses proteolysis (40), whereas IGF-I in combination with systemic amino acid infusion increases protein synthesis (41). Insulin appears to primarily inhibit proteolysis (42), but this effect is blunted by coadministration of GH (7). Taken together, it is uncertain to what degree IGF-I and insulin influenced protein metabolism in the present study, but neither of these factors has previously shown prominent effects on protein synthesis.
With GH therapy we observed unaltered insulin levels together with a significant increase in fasting glucose levels. GH has been shown to reduce peripheral glucose uptake and impair the ability of insulin to restrain hepatic glucose output (43, 44).The ability to maintain high glucose levels during fasting may be important because it prevents a rapid decline in glucose levels and reduces the need for providing gluconeogenic precursors from muscle protein.
The degree to which mobilization of lipids contributes to the anabolic action of GH has not been specifically investigated, but as the protein-sparing effect of lipid intermediates during fasting is well documented (39, 45), the increase in lipolysis and ketone bodies during GH treatment is a possible mechanism of substrate competition. The increased lipid oxidation could also be of importance for the insulin resistance seen because it may lead to a decreased glucose utilization during the "glucose-nonesterified fatty acids cycle " (46).
The observed GH-induced reduction in plasma cortisol levels is potentially interesting when considering the well-known protein catabolic effects of glucocorticoids. There is, however, evidence to suggest that GH primarily suppresses the levels of cortisol-binding globulin without altering free cortisol levels (47).
The present study adds new and supportive data to the concept of GH replacement in GH-deficient adults by demonstrating that hyposomatotropinemia during fasting accelerates protein loss. At the same time, it recapitulates and emphasizes the physiological role of GH as a regulator of substrate metabolism as originally hypothesized by Zierler and Rabinowitch (48) more than 30 yr ago.
Acknowledgments
We are grateful to Eva Seier Petersen and Lone Korsgård for excellent technical assistance. Dr. K. G. M. M. Alberti (University of Newcastle upon Tyne, Newcastle upon Tyne, UK) kindly provided the measurements of glycerol and 3-hydroxybutyrate.
Footnotes
1 Supported by Danish Research Council Grant 9600822 (to Århus
University-Novo Nordisk Center for Research in Growth and
Regeneration). 
Received October 18, 2000.
Revised January 24, 2001.
Accepted March 16, 2001.
References
This article has been cited by other articles:
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  H. Norrelund, C. Djurhuus, J. O. L. Jorgensen, S. Nielsen, K. S. Nair, O. Schmitz, J. S. Christiansen, and N. Moller Effects of GH on urea, glucose and lipid metabolism, and insulin sensitivity during fasting in GH-deficient patients Am J Physiol Endocrinol Metab, October 1, 2003; 285(4): E737 - E743. [Abstract] [Full Text] [PDF]
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 H. Norrelund, K. S. Nair, S. Nielsen, J. Frystyk, P. Ivarsen, J. O. L. Jorgensen, J. S. Christiansen, and N. Moller The Decisive Role of Free Fatty Acids for Protein Conservation during Fasting in Humans with and without Growth Hormone J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4371 - 4378. [Abstract] [Full Text] [PDF]
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