Growth
Hormone Improves Muscle Protein Metabolism and Whole Nitrogen Economy in Man During a Hyponitrogenous Diet
Stefan Lundeberg, Mins Belfrage, Jan Wernerman,
Body
Alexandra von der Decken, Stig Thunell, and Erik Vinnars
Healthy male volunteers (n = 12) were given a normocaloric hyponitrogenous diet for a conditioning period of 7 days. Thereafter they were blindly randomized to receive daily injections of methionyl recombinant human growth hormone (met-hGH) 0.06 IV/kg or saline during a second week of hyponitrogenous nutrition. The met-hGH group showed a lower urinary urea excretion and a lower serum concentration of urea as compared with the control group. In skeletal muscle, the polyribosome concentration, indicative of muscle protein synthesis, as well as the concentrations of glutamine, alanine, aspartate, serine, and threonine, decreased in the control group, whereas no such changes were seen in the met-hGH-treated group. Since provision of met-hGH prevented protein catabolism in muscle and improved whole body nitrogen economy, investigations of the possible beneficial effects of met-hGH to prevent skeletal muscle vast after surgical trauma are advocated. Copyriiht fi?1991 by W.B. Saunders Company
F
OLLOWING SURGICAL trauma, an obligatory loss of protein emerges.’ Whole body nitrogen balance becomes negative, and in skeletal muscle, the level of free glutamine declines and muscle protein synthesis is Iow.‘,~ Thz muscle protein catabolism can be counteracted by minimizing the traumatic stress”.” or by postoperative nutrition, including glutamine or cy-ketoglutarate.‘.’ However, conventional total parenteral nutrition (TPN) does not prevent muscle protein catabolism, even when unphysiologically high amounts of nitrogen are given.‘,’ Since the strategies to prevent losses of muscle protein described above may not always be suitable, administration of synthetic human growth hormone (hGH) may provide an alternative. A nitrogen-sparing effect is seen when hGH is administered together with hypocaloric intravenous feeding to healthy volunteers”’ or after surgery.” hGH also improves nitrogen economy during TPN in burn patients’* and following surgical trauma.” Since the improvement in nitrogen economy is accompanied by a retention of phosphorus and potassium, an advantageous effect on muscle protein metabolism can be expected.“’ The present investigation addressed the question if methionyl recombinant hGH (met-hGH) administered to healthy subjects on a normocaloric hyponitrogenous diet could spare muscle protein and improve whole body nitrogen economy. Muscle protein metabolism was studied by the use of two techniques based on completely different underlying assumptions. Thus, the concentrations of Free amino acids have been shown to exhibit highly reproducible changes in association with protein catabolism caused by malnutrition,‘4~‘” starvation,‘(’ semistarvation,” or trauma.’ The ribosome content and the size distribution of the ribosome particles give qualitative information about the state of muscle protein synthesis,‘” and the capacity for protein synthesis, as well as the synthesis activity, is assessed.‘” Both techniques have been used in numerous investigations to study muscle protein metabolism either as single assessment or in combination. After a conditioning period of 1 week on the hyponitrogenous diet, volunteers were blindly randomized to have a daily injection of met-hGH or saline. met-hGH was shown to cmprove the whole body nitrogen economy and to prevent a decline in the content of ribosomes and free amino acids in skeletal muscle. Metsbolism,
Vol 40, No 3 (March),
1991:
pp 315.322
MATERIALS
AND METHODS
Healthy male volunteers (n = 12) without medication and with normal medical history and physical examination participated in the study. They were randomized into two groups of six subjects each to be studied during a 2-week period. The groups did not differ concerning age, weight, or height (Table 1). During the trial, all subjects received a normocaloric and hyponitrogenous oral diet. The intake of nonprotein calories was 168 kJ/kg body weight (BW) per 24 hours, consisting of 50% fat and 50% carbohydrate. The protein intake was 0.3 g/kg BW per 24 hours (0.05 g N/kg BW/24 h). The food was given as three meals per day. The subjects were allowed free noncaloric fluid intake. The food regimen was designed by a dietitian and produced by the hospital kitchen. Urine was collected in 24-hour portions starting at 8:00 AM. The collection vessels contained 100 mL of 1 mol/L hydrochloric acid. During the first week of the study, the conditioning period, no injections were given. During the second week, the treatment period. one group of subjects received a daily intramuscular injection of met-hGH (Somatonorm, KabiVitrum. Stockholm, Sweden) 0.06 IUikg BW, the other saline. The injections were given in the large gluteal muscle after blood samples had been taken. One week in advance of and during the study period, normal daily activities were encouraged except for excessive physical activity. The nature, purposes, and potential risks of the experimental procedures were explained to the subjects before obtaining their voluntary consent. The study protocol was approved by the Ethics Committee of the Karolinska Institute, Stockholm, Sweden. Venous blood samples were taken each morning at 8:00 after an overnight fast. The serum concentrations of glucose (GOD-PAP, Boehringer Mannheim, Mannheim, Germany), free fatty acids,“’ triglycerides,” urea (Urease enzymatic UV test, Boehringer Mannheim), insulin (radioimmunoassay [RIA], Amersham. Cardiff, UK), and C-peptide (RIA. Hoechst, Frankfurt, Germany) were determined. Urine was analyzed for the excretion of urea and
From the Departments of Anesthesiology and Intensive Care. St Goran’s Hospital and Dandetyd S Hospital, Karolinska btstitute; the Department of Clinical Chemistry, St Gorank Hospital: and the Wenner-Gren Institute for Experimental Biology. Universin/ of Stockholm, Stockholm, Sweden. Supported by grants from the Swedish Medical Research Councd (Project No. 04210). the Maud and Bigger Gustavsson Foundation, and the County Council of Stockholm. Address reprint requests to Jan Wemenan, MD, PhD, Department of Anesthesiology and Intensive Care, St Goran ‘s Hospital, S-l 12 81 Stockholm, Sweden. Copyright 0 1991 by WB. Saunders Company 0026049.~/9114003-0017$03.0010 315
316
LUNDEBERG ET AL
Table 1. Characteristics Hyponitrogenous
of Healthy Male Volunteers Given a
Oral Diet for 2 Weeks, Who Were Blindly
Randomized to Receive Daily Injections of met-hGH (n = 6) or Saline (n = 6) During the Second Week met-hGH
Age tvr)
Group
Control Group
27.3 f 1.7
26.7 ? 1.9
181 + 1
186 ‘- 3
Before
74.7 -c 0.9
77.5 & 4.0
After
73.3 * 0.7
75.3 2 3.9
Height (cm) Weight (kg)
NOTE. Values are means 2 SEM.
ammonia (GLDH, kinetic UV tests, Merck, Darmstadt, Germany), creatinine (Jaffe reaction, Beckman ASTRA, Palo Alto, CA), and catecholamines.** Muscle biopsies were taken at the start of the investigation (day 0), after the conditioning period (day 7), and after the treatment period (day 15). The samples were taken from the lateral portion of the quadriceps femoris muscle approximately 15 cm above the knee. The percutaneous needle biopsy technique was used after giving local anesthesia confined to the skin and muscle fascia.23 Tissue aliquots were saved for analysis of ribosomes and free amino acids. The individual specimens were weighed three times during a 20-second period on an automated electrobalance (Cahn Instruments, Cerato, CA) and the original wet weight (ww) at time zero was extrapolated. All samples were freezed in liquid nitrogen within 5 minutes and thereafter stored at -80°C pending analysis. The specimens saved for ribosome determination were not stored for more than 4 weeks. The procedure for ribosome determination has been described in detail elsewhere.18 Thus, all specimens from the same subject were analyzed in the same run. In short, the tissue sample of approximately 60 mg was homogenized in a sucrose-containing medium with an RNAse inhibitor added. After a low-speed centrifugation at 2,500 x g for 15 minutes, the pellet containing nuclei, mitochondria, and membranes was saved for DNA analysis. The supernatant containing more than 80% of the cytoplasmatic RNA of the tissue was ultracentrifuged at 102,000 x g for 2 hours, assembling the ribosome particles in the pellet. After resuspension of the ribosome pellet, one aliquot was used for total ribosome determination by use of the absorbance at 260 nm. Another aliquot was layered onto a sucrose density gradient between 0.4 and 1.5 mol/L. After ultracentrifugation in a swing-out rotor at 149,000 X g for 1 hour, the gradient was pumped through a flow curvette and read continuously at 260 nm. In the ribosome profile obtained, the peaks corresponding to the 80s monoribosomes, the 40s and 60s ribosome subunits, and the polyribosomes were identified. The relative proportion of polyribosomes out of total ribosomes was calculated. The coefficient of variation is below 7% for the determination of the total ribosome content, as well as for the
relative proportion of polyribosomes. The DNA content of the pellet after the first centrifugation was determined by a fluorometric method using salmon DNA as a standard.” The tissue specimens of approximately 25 mg used for free amino acid determination were homogenized in sulfosalicylic acid, containing norleucine as internal standard.25 Also, plasma was precipitated in sulfosalicylic acid with addition of norleucine. After centrifugation, the supernatant was run through an ion-exchange chromatography automated amino acid analyzer (AlphaPlus, LKB, Bromma, Sweden) using lithium citrate buffers and DG-6 resin (Durum, Interaction, CA). Quantification was performed spectrophotometrically at 570 nm after reaction with ninhydrine at 135°C. The values given rely on samples taken in duplicate. The coefficients of variation for individual amino acids were between 4% and 9%. All values are given as means 2 SEM. The Wilcoxon matchedpair test and the Student’s t test for paired samples were used to compare observations within the groups, while Student’s t test for unpaired samples was used for between-group comparisons.‘”
RESULTS
male volunteers received a hyponitrogenous oral diet for a conditioning period of 1 week. They were then divided into two groups to receive daily injections of met-hGH or saline for an additional week. Daily blood samples and muscle biopsies were taken on days 0, 7, and 15, and urine was collected in 24-hour portions during the entire study period. Healthy
Urinary Analyses
The creatinine excretion was constant throughout the investigation, while the urinary urea excretion, in absolute values, as well as in relation to the creatinine excretion, was lower in both groups during the treatment period as compared with the conditioning period (P < .05; Table 2). The decrease was more pronounced in the met-hGH group as compared with the control group (P < .Ol; Fig 1). The ammonia excretion was unchanged in the met-hGH group, whereas the controls exhibited a lower excretion during the second week as compared with the first (P < .Ol). The noradrenaline content of urine was unaltered in both groups, while the adrenaline excretion declined in the met-hGH group, as well as in the control group, during the treatment period as compared with the conditioning period (P < .05). Among the variables analyzed in urine, no significant differences were observed between the tivo groups during the conditioning week.
Table 2. Urine Analyses met-hGH Day 1
Creatinine (mmol/d)
15.7 lr 0.9
Group
Week 1
15.3 + 0.7
Urea (mmol/d)
355 2 59
213 + 16
Ammonia (mmol/d)
39.0 + 5.0
27.4 -t 2.2
Noradrenaline (nmol/d) Adrenaline (nmol/d)
271 + 13
283 & 17
68.2 + 10.3
62.9 + 13.3
Control Group Week 2
15.0 + 0.6 101 f 14*1 26.6 + 0.9 308 + 28 39.6 + 10.7t
Day 1
Week 1
13.3 2 1.8
14.1 + 1.8
Week 2
13.4 r 1.2
298 & 47
169 + 28
122 ? 18’
30.0 * 3.0
23.0 + 2.1
18.7 2 1.77
240 + 44
289 2 61
260 ? 53
65.7 + 9.8
70.1 ? 9.7
45.0 2 7.0*
NOTE. All parameters were determined daily in the 24-hour urine collection given as means of weeks. Significantly different from the conditioning period: *P < .05,t P < .Ol, SP < ,001. Significantly different alteration between the 2 weeks as compared with the control group: §P < .Ol.
317
GROWTH HORMONE AND MUSCLE PROTEIN METABOLISM
Table 4. Ribosome Analysis in Skeletal Muscle met-hGHGroup
ControlGroup
Total ribosome concentration (OD: U/mg DNA) 20 *Treatment
period-,
Day 0
24.4 + 2.6
27.9 + 4.8
Day 7
20.3 2 2.8
19.1 2 1.3
Day 15
21.8 -c 1.8
18.7 + 1.8
Percentage proportion of polyribosomes out of total ribosomes 10
Day 0
53.2 2 2.9
Day 7
53.9 * 3.4
45.7 * 2.1 46.0 t 2.7
Day 15
52.2 + 3.6
37.8 t 2.4
NOTE. Values are means -C SEM.
0 0
Day
of
r
I
7
14
the
age proportion of polyribosomes out of total ribosomes did not change significantly in any of the groups during the study period (Table 4). When the concentration of polyribosomes per milligram of DNA was calculated by multiplying the two variables measured, a decrease was observed in the control group on day 15 as compared with day 0 (P < .Ol; Fig 2). In the met-hGH group, on the other hand, no such decline was seen. When the results were calculated for the two groups combined during the conditioning period, the total ribosome concentration declined by 19.7% t 7.8% (P < .05) and the polyribosome concentration by 20.9% 2 6.3% (P < .Ol).
Study
Fig 1. The daily excretion of urea per mmol creatinine in healthy subjects given a hyponitrogenous oral diet for 2 weeks. During the second week, subjects were blindly randomized to receive daily injections of met-hGH (n = 6; 0) or saline (n = 6; 0). Means ? SEM.
Serum Analyses
During the first week without met-hGH injections, there were significantly lower levels in the postabsorptive state of insulin, C-peptide, and triglycerides in the met-hGH group as compared with the control group (P < .OS;Table 3). The serum concentrations of glucose, triglycerides, and free fatty acids after an overnight fast were constant in both groups during the entire study. The insulin and C-peptide levels both increased in the met-hGH group during the treatment week as compared with the conditioning week (P : .0.5). These two variables were unaltered in the control group. The change in the met-hGH group was significantly different from the unaltered state of the control group (P < .OS). Serum urea concentration decreased in both groups during the second week as comparsd with the first (P < .05). The change was more pronounced among the met-hGH-treated subjects as compared with the controls (P < .Ol).
Amino Acid Analyses
The concentrations of free amino acids in muscle are given in Table 5. During the conditioning period, no significant changes were observed in any individual amino acid in either of the two groups. When the two groups were combined, an increase was seen for asparagine (P < .05). On day 15, after the treatment period, the muscle levels of glutamine, alanine, asparagine, threonine, and serine, as well as the total sum of amino acids and the sum of nonessential amino acids, were lower in the control group as compared with the met-hGH group (P < .05). In addition, the levels of alanine, leucine, phenylalanine, and carnosine, together with the sum of aromatic amino acids, declined in the control group during the second week (P < .05). In the met-hGH group, similar decreases were seen for leucine, valine, and the sum of the branched-chain amino acids, while the level of arginine increased (P < .05). For aspartate, threonine, serine, alanine. phenylalanine, arginine, and the sum of aromatic amino acids, the alter-
Ribosome Analyses
The total ribosome concentration expressed as optical density (OD) units per milligram of DNA and the percent-
Table 3. Serum Analyses met-hGHGroup Day 1
ControlGroup
Week 1
Week 2
Crea (mmol/L)
5.83 2 0.85
3.39 + 0.10
1.79 + 0.12t4
Insulin (mu/L)
7.25 t 1.20
8.33 + 0.30
9.49 + 0.50**
C-peptide (pg/L)
1.55 + 0.03
1.54 + 0.04
Glucose (mmol/L)
4.63 2 0.19
Triglycerides (mmol/L) Free fatty acids (pmol/L)
Day 1 5.08 ? 0.54
Week 1
Week 2
2.94 + 0.08
2.47 t- 0.03*
1.67
10.18 lr 0.47
9.43 + 0.37
1.80 I 0.07’$§
1.81 t 0.10
1.85 * 0.07
1.60 + 0.02
4.62 2 0.04
4.58 2 0.07
4.58 ? 0.14
4.62 k 0.05
4.54 + 0.07
0.81 + 0.09
0.75 -t 0.07
0.82 2 0.08
1.28?
1.07 5 0.08
1.15 5 0.12
616 2 83
811 2 160
539 f 58
556 2 35
478 + 35
12.00?
0.10
423 + 92
NOTE. The parameters were determined daily, but here the results are given as means of weeks. Significantly different from the conditioning period: *P < .05, tP < .Ol. Significantly different alteration between the 2 weeks as compared with the control group: SP < .05, §P < .Ol.
LUNDEBERG ET AL
+-Treatment 0
7
ation during the second week were significantly different between the two groups (P < .05). The concentrations of free amino acids in plasma are given in Table 6. During the conditioning period, the nonessential amino acids increased as compared with the initial values (P < .Ol), while the branched-chain amino acids declined (P < .OOl). The decrease of the branchedchain amino acids and the augmentation of the sum of the nonessential amino acids continued during the second week in the met-hGH group (P < .Ol), but not among the controls. For the nonessential amino acids, as well as the total sum of amino acids, the alteration during the second week of the study were significantly different between the two groups (P < .Ol).
period -
15
Day of the
Study
DISCUSSION
The effect of met-hGH on human skeletal muscle during hyponitrogenous feeding was evaluated by determination of free amino acids and ribosomes. Muscle protein synthesis as reflected by the polyribosome concentration declined after 1 week of low nitrogen intake, despite an adequate caloric intake. Addition of met-hGH normalized the polyribosome content, which was still diminished among the controls (Fig 2). One week of the nitrogen-deficient diet did
Fig 2. The concentration of polyribosomes per mg of DNA in healthy subjects given a hyponitrogenous oral diet for 2 weeks. During the second week, subjects were blindly randomized to receive daily injections of met-hGH (n = 6; 0) or saline (n = 6; 0). In the combined group, the values decreased during the conditioning period as compared with the initial values of 100% (P < .Ol). The values of the control group were low after the 2-week period as compared with the basal values (P < .Ol), whereas values for the met-hGH group were not. Means -+ SEM.
Table 5. Concentrations
Day0 Taurine
of Free Amino Acids in Muscle
Day 7
met-hGH Day15
Controls Day 15
12.20 r 1.31
13.14 r 1.05
11.77 k 1.26
Aspartate
1.02 + 0.09
1.06 + 0.08
1.23 + 0.13
0.84 + 0.07§//
Threonine
0.60 + 0.06
0.69 + 0.05
0.68 + 0.04
0.51 2 0.038/1
Serine
0.70 + 0.07
0.76 k 0.05
0.80 2 0.04
0.60 t 0.07811
Asparagine
0.34 + 0.05
0.49 ‘- 0.06*
0.47 + 0.06
0.29 + 0.03
10.27 + 1.23
Glutamate
2.59 ? 0.21
3.12 f 0.39
2.98 + 0.31
2.20 + 0.21
Glutamine
13.88 + 0.76
14.75 k 1.30
14.85 + 1.04
11 .oo k 0.481
Glycine
1.36 + 0.11
1.50 + 0.18
1.33 t 0.17
1.24 2 0.15
Alanine
2.40 k 0.21
2.85 k 0.23
3.29 2 0.21
2.29 k o.l3tll#
Valine
0.32 2 0.02
0.28 L 0.02
0.25 k 0.02*
0.21 + 0.02
lsoleucine
0.14 k 0.01
0.15 k 0.01
0.14 + 0.01
0.13 k 0.01
Leucine
0.23 k 0.02
0.24 + 0.02
0.22 k 0.02t
0.17 + 0.01t
Tyrosine
0.10 k 0.01
0.10 k 0.01
0.10 rf: 0.01
0.07 + 0.01
Phe
0.08 2 0.01
0.08 2 0.01
0.08 -t 0.01
0.05 2 0.01 q
Ornithine
0.25 + 0.03
0.21 + 0.01
0.21 + 0.02
0.17 2 0.02
Lysine
0.59 + 0.06
0.64 + 0.05
0.65 k 0.07
0.54 + 0.06
Histidine
0.38 + 0.04
0.40 + 0.03
0.40 k 0.06
0.32 t 0.03
Carnosine
4.90 ‘- 0.66
5.56 2 0.47
5.26 k 0.62
4.82 + 0.34t
Arginine
0.32 2 0.04
0.29 + 0.03
0.37 + 0.04t
0.27 2 0.03(1
Total AA
25.28 2 1.86
27.47 2 2.16
28.08 i 1.88
20.80 ‘- 1.055
Noness AA
19.10 ‘- 0.965
23.20 k 1.71
25.29 2 2.08
25.93 k 1.76
Ess AA
2.08 -c 0.17
2.18 2 0.13
2.15 2 0.16
1.70 t 0.13
BCAA
0.69 + 0.05
0.64 ? 0.04
0.61 + 0.05t
0.50 k 0.04t
Aromatic AA
0.19 + 0.02
0.18 ? 0.01
0.17 + 0.02
0.12 2 o.o1t//
Basic AA
1.38 2 0.21
1.30 k 0.09
1.42 2 0.13
1.08 t- 0.12
NOTE. Values are means k SEM. Abbreviations: AA, amino acids; Ess, essential; BCAA, branched chain amino acids. *Significantly differentfrom
the paired values of day 0 (P < ,051.
tSSignificantly different from the paired values of day 7 (P < .05, P < .Ol, respectively). ll§Significantly different on day 15 as compared with the met-hGH group (P < .05, P < .Ol, respectively). /I#Significantly different alteration between days 7 and 15 as compared with the met-hGH group (P < .05, P < .Ol, respectively).
319
GROWTH HORMONE AND MUSCLE PROTEIN METABOLISM
Table 6. Concentrations
-
of Free Amino Acids in Plasma met-hGH
Day 0
Day 7
Day 15
Controls Day 15
-
63 k 6
69 + 2
66 t 5
Aspartate
Taurine
5+0
520
621
Threonine
158 -+ 8
192 2 lot
192 + 15
17120
Serine
152 _t 9
78 _+ 6
521
135 + 7
168 f 9t
192 2 17
Asparagine
83 2 3
104 t 4*
120 k 18
95 IL 5
Glutamate
26 it 4
32 + 5
40 + 9
25 + 3
Glutamine
694 + 20
761 + 26t
892 + 7211
803 k 35**
Glycine
279 2 17
368 k 20*
387 f 37
387 k 29
Alanine
370 2 25
500 + 40t
706 2 425
511 r 52$*
48 + 3
41 +- 2*
41 -+ 4
313 k 8
247 + 9*
210 -t loll
Citrulline Valine
43 2 4 222 + 1ott
Cystine
21 2 3
20 -t 2
14 -c 2
19 k 2
Methionine
40 _f 3
43 k 3
50 -c 2
34 2 2**
lsoleucine Leucine
99 t 2
88 k 3t
188 ;t 5
164 ‘- 5t
87 + 4
83 2 5
147 t 711
148 2 7
Tyrosine
76 k 4
70 r 3
65 f 21
Phenylalanine
70 + 2
71 _f 2
64 + 35
63 i
Ornithine
67 -+ 7
57 k 3
56 k 4
67 t 7
179 k 91
210 2 12
Lysine
189 r 8
Histidine
87 + 4
Arginine
106 + 10
211 +8 83 + 3 99 + 10
59 + 39 1
74 + 3n
85 i 2#
102 z 16
102 2 85tt
Total AA
3,080 + 97
3,362 t 109”
3,647 t 2390
3,300 +- 114**
Noness AA
1,927 + 72
2,256 r 92t
2,641 + 21 Ill
2,292 f 8855
Ess AA
1,153 +- 30
1,106 t- 30
1,006 2 441
1,008 t 37 453 f 20
BCAA Aromatic
AA
Basic AA
600 + 12
499 + 15$
444 -+ i9n
146 2 6
141 -c4
128 2 48
121 *4
381 + 19
394 k 16
354 t 22
397 t 20
NOTE. Values are means r SEM. *tSSignificantly
different from the paired values of day 0 (P < .05, P < .Ol, P < ,001. respectively).
§ll ISignificantly
different from the paired values of day 7 (P < .05, P < .Ol, P < ,001, respectively).
#‘*Significantly tt$S§§Significantly
different
on day 15 as compared
different
alteration
with the met-hGH group (P < .05, P < ,001, respectively).
from the paired values on day 7 as compared
with the met-hGH
group
(P i
.05, P -. .Ol, P c ,001,
respectively).
influence the free amino acid content in muscle, but after a second week of low nitrogen intake, the levels of several individual amino acids, as well as the total sum of amino acids, declined. This depiction was not seen in the met-hGH group, except for the branched-chain amino acids. Ribosomc analysis is a qualitative estimate of protein synthesis.‘” The technique has been validated in human skeletal muscle under a variety of physiological and clinical situ;,ti ens. ?.‘~ ‘.i”.?7Considerable interindividual differences in the absolute values of ribosome parameters, as well as the magnitude of the changes in these parameters, are seen in connection with protein catabolism. Here, the total concentration of ribosomes decreased during the conditioning period. The decrease of the mean value in the control group was 32%. as compared with 17% in the group later receiving met-hGH treatment. This difference statistically insignificant. When interpretating ribosome analysis, significanr differences between paired samples reflect changes in protein synthesis. It is not advisable to draw conclusions from the magnitude of these changes without great caution.“,‘” When the concentration of polyribosomes was calculated. a reduction was seen after the conditioning not
period. After the treatment period, this reduction persisted in the control group, while the polyribosome content was normalized in the met-hGH group. Short-term starvation depresses muscle protein synthesis in man.‘x,2y Two days of refeeding following 3 days of starvation does not normalize the decrease in ribosome content, but the proportion of polyribosomes is restored.” In the present study, 1 week of met-hGH treatment prevented a decrease in the polyribosome concentration. Among free amino acids of muscle, the decline in the glutamine level is a hallmark of protein catabolism during starvation,16 as in several other muscle protein catabolic states.‘.” Malnutrition states, as represented by patients with anorexia nervosa” or gastric cancer,” show a similar glutamine depletion. After surgical trauma there is a correlation between the decrease in muscle glutamine content and the change in polyribosome concentration,” suggesting a close relationship between glutamine metabolism and muscle protein synthesis.“’ Here, the controls exhibited a decrease in muscle glutamine during the second week of the study, which the met-hGH group did not. On the contrary, plasma glutamine concentration became elevated in both groups, but the increase was most pronounced
320
in the met-hGH group. The increase in the branched-chain amino acids and aromatic amino acids seen after short-term starvation or following surgical trauma was not evident here. On the contrary, a decline of these amino acids in muscle, as well as in plasma, was recorded in muscle irrespective of whether met-hGH was provided or not. Malnutrition is not associated with low levels of branchedchain amino acids. On the other hand, uremic patients, as well as patients with liver insufficiency, often show low values.31 In protein catabolic states, the net flux of free amino acids from the periphery to the splanchnic area increase.” It is not settled whether this transport of amino acids results from a “spill over” in the periphery or from a “suction” in the splanchnic area. It is also unclear what mechanisms regulate such changes in amino acid metabolism and transport. Elevated efflux of amino acids from muscle is accompanied by low levels of free amino acids in the tissue. As described above, such changes are most pronounced for glutamine and other nonessential amino acids, while the essential amino acids stay unaltered or increase in concentration.2.*.14,15~17,28 Here, the normalization of the muscle amino acids in the met-hGH group is indicative of nearnormal amino acid and protein metabolism. This possible effect of met-hGH treatment may be exerted in muscle tissue and/or in the splanchnic area. Obviously, the utilization of amino acids for urea production is low under the influence of met-hGH, but this may be related to the magnitude of the amino acid supply, as well as to other regulative factors. Nitrogen economy is improved when hGH is administered to healthy subjects or patients on a hypocaloric diet.“‘,” Here, an adequate caloric intake was given in combination with an insufficient amount of nitrogen. The first week was a conditioning period for the volunteers to approach a metabolic steady-state and to become comparable during the second week when they were randomized to receive met-hGH or not. Although the characteristics of the volunteers were comparable, some biochemical parameters showed differences during the first week. These dissimilarities, which are likely to be attributable to the comparatively small number of subjects included in each group, were diminished when entering the treatment period during the second week of the study. The delayed influence on muscle amino acids of the nitrogen-deficient diet, and the comparatively slow adjustment of the urinary urea excretion, contrast to the more rapid adaptation seen during total starvation.33 The mechanism of the nitrogen-saving effect of hGH is unclear. The urinary urea excretion is attenuated in association with surgical trauma and burns.“.” This is the case during circumstances where adequate support of both calories and nitrogen is given,‘2213as well as where hypocaloric and/or hyponitrogenous regimens are applied.” During starvation, a more efficient nitrogen utilization is observed in normal-weight,‘0 but not in obese subjects.34 A major part of the hGH effect on whole body nitrogen economy is mediated through urea metabolism.35 In the present work, a diminished urea excretion was accompa-
LUNDEBERG ET AL
nied by a low urea concentration in serum. hGH effects the release of both glucagon and insulin from pancreas,36 and glucagon is the major determinant of urea production3’ Unfortunately, glucagon was not measured in the present investigation, but the augmented levels of insulin and C-peptide were reproduced.“’ No effect on serum glucose in the postabsorptive state was seen. The daily urea excretion declined during the conditioning period to become adapted to an approximately steadystate during the second week (Fig 1). The adaption was quicker in the met-hGH group than in the control group. The differences between the two groups in urea and creatinine excretion are in accord with the literature.10,35 The excretion of ammonia decreased during the second week in the control group. No changes in urinary ammonia are reported in association with hGH treatment.‘0.12X34 In sepsis and after surgical trauma, an increase of the hGH level in serum is seen,35.38which is abolished by spinal anesthesia blocking the response of stress hormones after surgical trauma.3S High hGH levels are associated with elevated levels of corticotropin and corticosteroids,38 but catecholamines do not augment hGH.39 Suppression of hGH sometimes noted in septic subjects is associated with a high mortality.40 This pathophysiological increment of hGH has been suggested to be important for the regulation of protein metabolism.41 Acromegaly in man and pathological overproduction of GH in animals are accompanied by a hypertrophy of skeletal muscle.42 Whole body protein synthesis, as well as whole body protein degradation, is enhanced postoperatively when met-hGH is provided.” However, the role of an unphysiologically high hGH level on human muscle protein metabolism after trauma is not clarified. The results presented here show that a comparatively low dose of met-hGH administered to healthy subjects affects muscle protein metabolism. In addition, the techniques used provided information about the state of muscle protein metabolism. The ribosome technique is an in vivo method that allows repetitive measurements in a patient, to monitor the course of changes in muscle protein synthesis.3*6,43 Studies evaluating the effects of met-hGH on skeletal muscle after elective surgery or in intensive care patients should include several techniques in parallel to assess protein metabolism, resting on different underlying assumptions. To conclude, changes consistent with protein catabolism in skeletal muscle were prevented when met-hGH was provided to healthy subjects on a hyponitrogenous oral diet. Whole body nitrogen economy was improved as shown by a diminished urea excretion and a low serum urea level indicative of a decreased urea production. Investigations of the possible beneficial effect of met-hGH to prevent skeletal muscle vast after surgical trauma are advocated. ACKNOWLEDGMENT We are thankful for the skillful nursing assistance of E. Bostedt and the skillful technical assistance of C. Hebert, L. Thunblad, and Dr M.R. Ah, and to L. Nordhmd for the help in designing computer programs for the amino acid calculations. Somatonorm was generously supplied by KabiVitrum Peptide Hormones, Stockholm, Sweden.
GROWTH HORMONE
AND MUSCLE PROTEIN METABOLISM
321
REFERENCES 1. Kinney JM, Long CL, Gump FE, et al: Tissue composition weight loss in surgical patients. Ann Surg 168:459-474,196s
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2. Vinnars E, Bergstrom J, Ftirst P: Influence of the postoperative state on the intracellular free amino acids in human muscle tissue. Ann Surg 182:665-671.1975 3. Wernerman J. von der Decken A, Vinnars E: Protein synthesis i? skeletal muscle in relation to nitrogen balance after abdominal surgery. The effect of total parenteral nutrition. JPEN 10:578582. 1986 4. Brandt MR. Fernandes A, Moedhorst analgesia improves postoperative nitrogen 1:1106-1108, 1978
R, et al: Epidural balance. Br Med J
5. Christensen T, Waaben J, Lindeburg T, et al: Epidural analgesia attenuates postoperative changes in muscle amino acids. Acta Chir Stand 152:407-411,1986 6. Wernerman J, Hammarqvist F. von der Decken A, et al: Ornithine-alpha-ketoglutarate improves skeletal muscle protein syntnesis as assessed by ribosome analysis and nitrogen use after surgery. Ann Surg 206:674-678,1987 7. Hammarqvist F, Wernerman J, Ali MR, et al: Addition of glutamine to total parenteral nutrition after elective abdominal surgery spares free glutamine in muscle, counteracts the fall in muscle protein synthesis, and improves nitrogen balance. Ann Surg 209:455-461, 1989 8. Vinnars E, Holmstrom B, Schildt B, et al: Metabolic effects of four intravenous nutritional regimens in patients undergoing elective surgery. II. Muscle amino acids and energy-rich phosphates. Clin Nutr 2:3-11, 1983 9. Larsson requirements lY9C
J, Lennmarken C, MPrtensson J, et al: Nitrogen in severely injured patients. Br J Surg 77:413-416,
1C. Manson JM, Wilmore DW: Positive human growth hormone and hypocaloric Surgery 100:188-197.1986 11. Ponting GA. Halliday D. Teale JD, positive nitrogen balance with intravenous growth hormone. Lancet 1:438-440,1988
nitrogen balance with intravenous feeding. et al: Postoperative hyponutrition and
12. Wilmore DW, Moylan JA Jr, Bristow BF, et al: Anabolic effects of human growth hormone and high caloric feedings following thermal injury. Surg Gynecol Obstet 138:875-884, 1974 13. Ward HC, Halliday D, Sim AJW: Protein and energy metabolism with biosynthetic human growth hormone after gastrointestinal surgery. Ann Surg 206:56-61, 1987 14. Sandstedt S, Symreng T, Larsson J: Changes in muscle and plasma amino acid metabolism in severe malnutrition-The influence of total parenteral nutrition. Clin Nutr 4:13-19,198s 15. Sandstedt S, Lennmarken C, Symreng T. et al: The effect of pre-operative total parenteral nutrition on energy-rich phosphates, electrolytes and free amino acids in skeletal muscle of malnourished patients with gastric carcinoma. Br J Surg 72:920-924, 1985 16 Vinnars E, Bergstrom, Fiirst P: Effects of starvation on plasma and muscle amino acid concentration in normal subjects. Clin Nutr 6:62, 1987 (suppl) 17 Askanazi J, Elwyn DH, Kinney JM, et al: Muscle and plasma amino acids after injury: The role of inactivity. Ann Surg 188:797803, 1978 18 Wernerman tion of ribosomes during starvation.
J, von der Decken A, Vinnars E: Size distribuin biopsy specimens of human skeletal muscle Metabolism 34:665-669,1985
19 Wernerman J, von der Decken A,Vinnars E: The interpretation of ribosome determinations to assess protein synthesis in human skeletal muscle. Infusionstherapie 13:162-165, 1986
20. Ho RJ: Radiochemical assay of long chain fatty acids using “‘Ni as tracer. Anal Biochem 36:105-113, 1970 21. Fletcher MJ: A calorimetric method for estimating serum triglycerides. Clin Chem Acta 22:393-397, 1968 22. Riggin RM, Kissinger PT: Determination of catecholamines in urine by reverse-phase liquid chromatography with electrochemical detection. Anal Chem 49:2109-2111. 1977 23. Bergstrom J: Muscle electrolytes in man. Stand J Clin Lah Invest 14:11-13, 1962 (suppl 168) 24. Setaro F, Morely CBD: A modified fluorometric method for the determination of microgram quantities of DNA from cell or tissue cultures. Anal Biochem 71:313-317. 1976 25. Bergstrom J, Furst P, Noree L-O, et al: The intracellular free amino acid concentrations in human muscle tissue. J Appl Physiol36:690-697, 1974 26. Snedecor GW, Cochran Ames, IA, Iowa State University
WG: Statistical Methods Press, 1967, pp 91-119
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27. Wernerman J, von der Decken A, Vinnars E: Polyribosome concentration in human skeletal muscle after starvation and parenteral or enteral refeeding. Metabolism 35:447-451. 1986 28. Hammarqvist F: Intravenous amino acid supply after trauma: The effects of glutamine on muscle protein metabolism. Thesis. Karolinska Institute, Stockholm, Sweden, 1989 29. Es&P, McNurlan MA, Calder AC. et al: The etIect of total starvation on protein synthesis rate in human skeletal muscle determined by the large dose technique. Clin Nutr 8:102. 1989 (suppl) 30. MacLennan PA, Brown RA, Rennie MJ: A positive relationship between protein synthesis rate and intracellular glutamine concentration in perfused rat skeletal muscle. FEBS Lett 215:187191,1987 31. Bergstrom J. Filrst P. Noree L-O, et al: Intracellular free amino acids in muscle tissue of patients with chronic uremia: EfIect of peritoneal dialysis and infusion of essential amino acids. Clin Sci Mol Med 54:51-60,1978 32. Wernerman J. Vinnars E: The effect of trauma and surgery on interorgan fluxes of amino acids in man. Clin Sci 73:129-133, 1987 33. Giesecke K, Magnusson I, Ahlberg M. et al: Protein and amino acid metabolism during early starvation as reflected by excretion of urea and methylhistidines. Metabolism 38:1196-1200, 1989 34. Clemmons DR, Snyder DK, Williams R, et al: Growth hormone administration conserves lean body mass during dietary restriction in obese subjects. J Clin Endocrinol Metabol 64:878883,1987 35. Dahn MS, Lange P: Hormonal changes and their influence on metabolism and nutrition in critically ill. Intensive Care Med 8:209-213,1982 36. Vranic AS, Sisek OV, Pelicova Z: Effects of growth hormone on acute glucagon and insulin release. Am J Physiol 237:E107E112.1979 37. Wolfe BM, Culebras JM, Aoki TT, et al: The effects of glucagon on protein metabolism in normal man. Surgery X6:248256,1979 38. Newsome HH. Rose JC: The response of human adrenocorticotrophic hormone and growth hormone to surgical stress. J Clin Endocrinol Metabol33:481-487, 1971 39. Hanson EJ. Brennan MF. O’Connell RC. et al: Response of glucose, insulin, free fatty acid, and human growth hormone to norepinephrine and hemorrhage in normal man. Ann Surg 177:453457.1973 40. Dahn M, Bouwman DL. Kirkpatrick JR: The sepsis-glucose
322
intolerance riddle: A hormonal explanation. Surgery 86:423-428, 1979 41. Dahn M, Kirkpatrick JR, Bouwman DL: Sepsis, glucose intolerance and protein mahmtrions, a metabolic paradox. Arch Surg 115:1415-1418,198O 42. Riss TL, Novakofski J, Bechtel PJ: Skeletal muscle hypertro-
LUNDEBERG ET AL
phy in rats having growth hormone-secreting tumor. J Appl Physiol 61:1732-1735,1986 43. Petersson B, Wernerman J, Wailer S-O, et al: Elective surgery depresses muscle protein synthesis and augments subjective fatigue more than 30 days postoperatively. Br J Surg 77:796800,199O
Hormone Improves Muscle Protein Metabolism and Whole Nitrogen Economy in Man During a Hyponitrogenous Diet
Stefan Lundeberg, Mins Belfrage, Jan Wernerman,
Body
Alexandra von der Decken, Stig Thunell, and Erik Vinnars
Healthy male volunteers (n = 12) were given a normocaloric hyponitrogenous diet for a conditioning period of 7 days. Thereafter they were blindly randomized to receive daily injections of methionyl recombinant human growth hormone (met-hGH) 0.06 IV/kg or saline during a second week of hyponitrogenous nutrition. The met-hGH group showed a lower urinary urea excretion and a lower serum concentration of urea as compared with the control group. In skeletal muscle, the polyribosome concentration, indicative of muscle protein synthesis, as well as the concentrations of glutamine, alanine, aspartate, serine, and threonine, decreased in the control group, whereas no such changes were seen in the met-hGH-treated group. Since provision of met-hGH prevented protein catabolism in muscle and improved whole body nitrogen economy, investigations of the possible beneficial effects of met-hGH to prevent skeletal muscle vast after surgical trauma are advocated. Copyriiht fi?1991 by W.B. Saunders Company
F
OLLOWING SURGICAL trauma, an obligatory loss of protein emerges.’ Whole body nitrogen balance becomes negative, and in skeletal muscle, the level of free glutamine declines and muscle protein synthesis is Iow.‘,~ Thz muscle protein catabolism can be counteracted by minimizing the traumatic stress”.” or by postoperative nutrition, including glutamine or cy-ketoglutarate.‘.’ However, conventional total parenteral nutrition (TPN) does not prevent muscle protein catabolism, even when unphysiologically high amounts of nitrogen are given.‘,’ Since the strategies to prevent losses of muscle protein described above may not always be suitable, administration of synthetic human growth hormone (hGH) may provide an alternative. A nitrogen-sparing effect is seen when hGH is administered together with hypocaloric intravenous feeding to healthy volunteers”’ or after surgery.” hGH also improves nitrogen economy during TPN in burn patients’* and following surgical trauma.” Since the improvement in nitrogen economy is accompanied by a retention of phosphorus and potassium, an advantageous effect on muscle protein metabolism can be expected.“’ The present investigation addressed the question if methionyl recombinant hGH (met-hGH) administered to healthy subjects on a normocaloric hyponitrogenous diet could spare muscle protein and improve whole body nitrogen economy. Muscle protein metabolism was studied by the use of two techniques based on completely different underlying assumptions. Thus, the concentrations of Free amino acids have been shown to exhibit highly reproducible changes in association with protein catabolism caused by malnutrition,‘4~‘” starvation,‘(’ semistarvation,” or trauma.’ The ribosome content and the size distribution of the ribosome particles give qualitative information about the state of muscle protein synthesis,‘” and the capacity for protein synthesis, as well as the synthesis activity, is assessed.‘” Both techniques have been used in numerous investigations to study muscle protein metabolism either as single assessment or in combination. After a conditioning period of 1 week on the hyponitrogenous diet, volunteers were blindly randomized to have a daily injection of met-hGH or saline. met-hGH was shown to cmprove the whole body nitrogen economy and to prevent a decline in the content of ribosomes and free amino acids in skeletal muscle. Metsbolism,
Vol 40, No 3 (March),
1991:
pp 315.322
MATERIALS
AND METHODS
Healthy male volunteers (n = 12) without medication and with normal medical history and physical examination participated in the study. They were randomized into two groups of six subjects each to be studied during a 2-week period. The groups did not differ concerning age, weight, or height (Table 1). During the trial, all subjects received a normocaloric and hyponitrogenous oral diet. The intake of nonprotein calories was 168 kJ/kg body weight (BW) per 24 hours, consisting of 50% fat and 50% carbohydrate. The protein intake was 0.3 g/kg BW per 24 hours (0.05 g N/kg BW/24 h). The food was given as three meals per day. The subjects were allowed free noncaloric fluid intake. The food regimen was designed by a dietitian and produced by the hospital kitchen. Urine was collected in 24-hour portions starting at 8:00 AM. The collection vessels contained 100 mL of 1 mol/L hydrochloric acid. During the first week of the study, the conditioning period, no injections were given. During the second week, the treatment period. one group of subjects received a daily intramuscular injection of met-hGH (Somatonorm, KabiVitrum. Stockholm, Sweden) 0.06 IUikg BW, the other saline. The injections were given in the large gluteal muscle after blood samples had been taken. One week in advance of and during the study period, normal daily activities were encouraged except for excessive physical activity. The nature, purposes, and potential risks of the experimental procedures were explained to the subjects before obtaining their voluntary consent. The study protocol was approved by the Ethics Committee of the Karolinska Institute, Stockholm, Sweden. Venous blood samples were taken each morning at 8:00 after an overnight fast. The serum concentrations of glucose (GOD-PAP, Boehringer Mannheim, Mannheim, Germany), free fatty acids,“’ triglycerides,” urea (Urease enzymatic UV test, Boehringer Mannheim), insulin (radioimmunoassay [RIA], Amersham. Cardiff, UK), and C-peptide (RIA. Hoechst, Frankfurt, Germany) were determined. Urine was analyzed for the excretion of urea and
From the Departments of Anesthesiology and Intensive Care. St Goran’s Hospital and Dandetyd S Hospital, Karolinska btstitute; the Department of Clinical Chemistry, St Gorank Hospital: and the Wenner-Gren Institute for Experimental Biology. Universin/ of Stockholm, Stockholm, Sweden. Supported by grants from the Swedish Medical Research Councd (Project No. 04210). the Maud and Bigger Gustavsson Foundation, and the County Council of Stockholm. Address reprint requests to Jan Wemenan, MD, PhD, Department of Anesthesiology and Intensive Care, St Goran ‘s Hospital, S-l 12 81 Stockholm, Sweden. Copyright 0 1991 by WB. Saunders Company 0026049.~/9114003-0017$03.0010 315
316
LUNDEBERG ET AL
Table 1. Characteristics Hyponitrogenous
of Healthy Male Volunteers Given a
Oral Diet for 2 Weeks, Who Were Blindly
Randomized to Receive Daily Injections of met-hGH (n = 6) or Saline (n = 6) During the Second Week met-hGH
Age tvr)
Group
Control Group
27.3 f 1.7
26.7 ? 1.9
181 + 1
186 ‘- 3
Before
74.7 -c 0.9
77.5 & 4.0
After
73.3 * 0.7
75.3 2 3.9
Height (cm) Weight (kg)
NOTE. Values are means 2 SEM.
ammonia (GLDH, kinetic UV tests, Merck, Darmstadt, Germany), creatinine (Jaffe reaction, Beckman ASTRA, Palo Alto, CA), and catecholamines.** Muscle biopsies were taken at the start of the investigation (day 0), after the conditioning period (day 7), and after the treatment period (day 15). The samples were taken from the lateral portion of the quadriceps femoris muscle approximately 15 cm above the knee. The percutaneous needle biopsy technique was used after giving local anesthesia confined to the skin and muscle fascia.23 Tissue aliquots were saved for analysis of ribosomes and free amino acids. The individual specimens were weighed three times during a 20-second period on an automated electrobalance (Cahn Instruments, Cerato, CA) and the original wet weight (ww) at time zero was extrapolated. All samples were freezed in liquid nitrogen within 5 minutes and thereafter stored at -80°C pending analysis. The specimens saved for ribosome determination were not stored for more than 4 weeks. The procedure for ribosome determination has been described in detail elsewhere.18 Thus, all specimens from the same subject were analyzed in the same run. In short, the tissue sample of approximately 60 mg was homogenized in a sucrose-containing medium with an RNAse inhibitor added. After a low-speed centrifugation at 2,500 x g for 15 minutes, the pellet containing nuclei, mitochondria, and membranes was saved for DNA analysis. The supernatant containing more than 80% of the cytoplasmatic RNA of the tissue was ultracentrifuged at 102,000 x g for 2 hours, assembling the ribosome particles in the pellet. After resuspension of the ribosome pellet, one aliquot was used for total ribosome determination by use of the absorbance at 260 nm. Another aliquot was layered onto a sucrose density gradient between 0.4 and 1.5 mol/L. After ultracentrifugation in a swing-out rotor at 149,000 X g for 1 hour, the gradient was pumped through a flow curvette and read continuously at 260 nm. In the ribosome profile obtained, the peaks corresponding to the 80s monoribosomes, the 40s and 60s ribosome subunits, and the polyribosomes were identified. The relative proportion of polyribosomes out of total ribosomes was calculated. The coefficient of variation is below 7% for the determination of the total ribosome content, as well as for the
relative proportion of polyribosomes. The DNA content of the pellet after the first centrifugation was determined by a fluorometric method using salmon DNA as a standard.” The tissue specimens of approximately 25 mg used for free amino acid determination were homogenized in sulfosalicylic acid, containing norleucine as internal standard.25 Also, plasma was precipitated in sulfosalicylic acid with addition of norleucine. After centrifugation, the supernatant was run through an ion-exchange chromatography automated amino acid analyzer (AlphaPlus, LKB, Bromma, Sweden) using lithium citrate buffers and DG-6 resin (Durum, Interaction, CA). Quantification was performed spectrophotometrically at 570 nm after reaction with ninhydrine at 135°C. The values given rely on samples taken in duplicate. The coefficients of variation for individual amino acids were between 4% and 9%. All values are given as means 2 SEM. The Wilcoxon matchedpair test and the Student’s t test for paired samples were used to compare observations within the groups, while Student’s t test for unpaired samples was used for between-group comparisons.‘”
RESULTS
male volunteers received a hyponitrogenous oral diet for a conditioning period of 1 week. They were then divided into two groups to receive daily injections of met-hGH or saline for an additional week. Daily blood samples and muscle biopsies were taken on days 0, 7, and 15, and urine was collected in 24-hour portions during the entire study period. Healthy
Urinary Analyses
The creatinine excretion was constant throughout the investigation, while the urinary urea excretion, in absolute values, as well as in relation to the creatinine excretion, was lower in both groups during the treatment period as compared with the conditioning period (P < .05; Table 2). The decrease was more pronounced in the met-hGH group as compared with the control group (P < .Ol; Fig 1). The ammonia excretion was unchanged in the met-hGH group, whereas the controls exhibited a lower excretion during the second week as compared with the first (P < .Ol). The noradrenaline content of urine was unaltered in both groups, while the adrenaline excretion declined in the met-hGH group, as well as in the control group, during the treatment period as compared with the conditioning period (P < .05). Among the variables analyzed in urine, no significant differences were observed between the tivo groups during the conditioning week.
Table 2. Urine Analyses met-hGH Day 1
Creatinine (mmol/d)
15.7 lr 0.9
Group
Week 1
15.3 + 0.7
Urea (mmol/d)
355 2 59
213 + 16
Ammonia (mmol/d)
39.0 + 5.0
27.4 -t 2.2
Noradrenaline (nmol/d) Adrenaline (nmol/d)
271 + 13
283 & 17
68.2 + 10.3
62.9 + 13.3
Control Group Week 2
15.0 + 0.6 101 f 14*1 26.6 + 0.9 308 + 28 39.6 + 10.7t
Day 1
Week 1
13.3 2 1.8
14.1 + 1.8
Week 2
13.4 r 1.2
298 & 47
169 + 28
122 ? 18’
30.0 * 3.0
23.0 + 2.1
18.7 2 1.77
240 + 44
289 2 61
260 ? 53
65.7 + 9.8
70.1 ? 9.7
45.0 2 7.0*
NOTE. All parameters were determined daily in the 24-hour urine collection given as means of weeks. Significantly different from the conditioning period: *P < .05,t P < .Ol, SP < ,001. Significantly different alteration between the 2 weeks as compared with the control group: §P < .Ol.
317
GROWTH HORMONE AND MUSCLE PROTEIN METABOLISM
Table 4. Ribosome Analysis in Skeletal Muscle met-hGHGroup
ControlGroup
Total ribosome concentration (OD: U/mg DNA) 20 *Treatment
period-,
Day 0
24.4 + 2.6
27.9 + 4.8
Day 7
20.3 2 2.8
19.1 2 1.3
Day 15
21.8 -c 1.8
18.7 + 1.8
Percentage proportion of polyribosomes out of total ribosomes 10
Day 0
53.2 2 2.9
Day 7
53.9 * 3.4
45.7 * 2.1 46.0 t 2.7
Day 15
52.2 + 3.6
37.8 t 2.4
NOTE. Values are means -C SEM.
0 0
Day
of
r
I
7
14
the
age proportion of polyribosomes out of total ribosomes did not change significantly in any of the groups during the study period (Table 4). When the concentration of polyribosomes per milligram of DNA was calculated by multiplying the two variables measured, a decrease was observed in the control group on day 15 as compared with day 0 (P < .Ol; Fig 2). In the met-hGH group, on the other hand, no such decline was seen. When the results were calculated for the two groups combined during the conditioning period, the total ribosome concentration declined by 19.7% t 7.8% (P < .05) and the polyribosome concentration by 20.9% 2 6.3% (P < .Ol).
Study
Fig 1. The daily excretion of urea per mmol creatinine in healthy subjects given a hyponitrogenous oral diet for 2 weeks. During the second week, subjects were blindly randomized to receive daily injections of met-hGH (n = 6; 0) or saline (n = 6; 0). Means ? SEM.
Serum Analyses
During the first week without met-hGH injections, there were significantly lower levels in the postabsorptive state of insulin, C-peptide, and triglycerides in the met-hGH group as compared with the control group (P < .OS;Table 3). The serum concentrations of glucose, triglycerides, and free fatty acids after an overnight fast were constant in both groups during the entire study. The insulin and C-peptide levels both increased in the met-hGH group during the treatment week as compared with the conditioning week (P : .0.5). These two variables were unaltered in the control group. The change in the met-hGH group was significantly different from the unaltered state of the control group (P < .OS). Serum urea concentration decreased in both groups during the second week as comparsd with the first (P < .05). The change was more pronounced among the met-hGH-treated subjects as compared with the controls (P < .Ol).
Amino Acid Analyses
The concentrations of free amino acids in muscle are given in Table 5. During the conditioning period, no significant changes were observed in any individual amino acid in either of the two groups. When the two groups were combined, an increase was seen for asparagine (P < .05). On day 15, after the treatment period, the muscle levels of glutamine, alanine, asparagine, threonine, and serine, as well as the total sum of amino acids and the sum of nonessential amino acids, were lower in the control group as compared with the met-hGH group (P < .05). In addition, the levels of alanine, leucine, phenylalanine, and carnosine, together with the sum of aromatic amino acids, declined in the control group during the second week (P < .05). In the met-hGH group, similar decreases were seen for leucine, valine, and the sum of the branched-chain amino acids, while the level of arginine increased (P < .05). For aspartate, threonine, serine, alanine. phenylalanine, arginine, and the sum of aromatic amino acids, the alter-
Ribosome Analyses
The total ribosome concentration expressed as optical density (OD) units per milligram of DNA and the percent-
Table 3. Serum Analyses met-hGHGroup Day 1
ControlGroup
Week 1
Week 2
Crea (mmol/L)
5.83 2 0.85
3.39 + 0.10
1.79 + 0.12t4
Insulin (mu/L)
7.25 t 1.20
8.33 + 0.30
9.49 + 0.50**
C-peptide (pg/L)
1.55 + 0.03
1.54 + 0.04
Glucose (mmol/L)
4.63 2 0.19
Triglycerides (mmol/L) Free fatty acids (pmol/L)
Day 1 5.08 ? 0.54
Week 1
Week 2
2.94 + 0.08
2.47 t- 0.03*
1.67
10.18 lr 0.47
9.43 + 0.37
1.80 I 0.07’$§
1.81 t 0.10
1.85 * 0.07
1.60 + 0.02
4.62 2 0.04
4.58 2 0.07
4.58 ? 0.14
4.62 k 0.05
4.54 + 0.07
0.81 + 0.09
0.75 -t 0.07
0.82 2 0.08
1.28?
1.07 5 0.08
1.15 5 0.12
616 2 83
811 2 160
539 f 58
556 2 35
478 + 35
12.00?
0.10
423 + 92
NOTE. The parameters were determined daily, but here the results are given as means of weeks. Significantly different from the conditioning period: *P < .05, tP < .Ol. Significantly different alteration between the 2 weeks as compared with the control group: SP < .05, §P < .Ol.
LUNDEBERG ET AL
+-Treatment 0
7
ation during the second week were significantly different between the two groups (P < .05). The concentrations of free amino acids in plasma are given in Table 6. During the conditioning period, the nonessential amino acids increased as compared with the initial values (P < .Ol), while the branched-chain amino acids declined (P < .OOl). The decrease of the branchedchain amino acids and the augmentation of the sum of the nonessential amino acids continued during the second week in the met-hGH group (P < .Ol), but not among the controls. For the nonessential amino acids, as well as the total sum of amino acids, the alteration during the second week of the study were significantly different between the two groups (P < .Ol).
period -
15
Day of the
Study
DISCUSSION
The effect of met-hGH on human skeletal muscle during hyponitrogenous feeding was evaluated by determination of free amino acids and ribosomes. Muscle protein synthesis as reflected by the polyribosome concentration declined after 1 week of low nitrogen intake, despite an adequate caloric intake. Addition of met-hGH normalized the polyribosome content, which was still diminished among the controls (Fig 2). One week of the nitrogen-deficient diet did
Fig 2. The concentration of polyribosomes per mg of DNA in healthy subjects given a hyponitrogenous oral diet for 2 weeks. During the second week, subjects were blindly randomized to receive daily injections of met-hGH (n = 6; 0) or saline (n = 6; 0). In the combined group, the values decreased during the conditioning period as compared with the initial values of 100% (P < .Ol). The values of the control group were low after the 2-week period as compared with the basal values (P < .Ol), whereas values for the met-hGH group were not. Means -+ SEM.
Table 5. Concentrations
Day0 Taurine
of Free Amino Acids in Muscle
Day 7
met-hGH Day15
Controls Day 15
12.20 r 1.31
13.14 r 1.05
11.77 k 1.26
Aspartate
1.02 + 0.09
1.06 + 0.08
1.23 + 0.13
0.84 + 0.07§//
Threonine
0.60 + 0.06
0.69 + 0.05
0.68 + 0.04
0.51 2 0.038/1
Serine
0.70 + 0.07
0.76 k 0.05
0.80 2 0.04
0.60 t 0.07811
Asparagine
0.34 + 0.05
0.49 ‘- 0.06*
0.47 + 0.06
0.29 + 0.03
10.27 + 1.23
Glutamate
2.59 ? 0.21
3.12 f 0.39
2.98 + 0.31
2.20 + 0.21
Glutamine
13.88 + 0.76
14.75 k 1.30
14.85 + 1.04
11 .oo k 0.481
Glycine
1.36 + 0.11
1.50 + 0.18
1.33 t 0.17
1.24 2 0.15
Alanine
2.40 k 0.21
2.85 k 0.23
3.29 2 0.21
2.29 k o.l3tll#
Valine
0.32 2 0.02
0.28 L 0.02
0.25 k 0.02*
0.21 + 0.02
lsoleucine
0.14 k 0.01
0.15 k 0.01
0.14 + 0.01
0.13 k 0.01
Leucine
0.23 k 0.02
0.24 + 0.02
0.22 k 0.02t
0.17 + 0.01t
Tyrosine
0.10 k 0.01
0.10 k 0.01
0.10 rf: 0.01
0.07 + 0.01
Phe
0.08 2 0.01
0.08 2 0.01
0.08 -t 0.01
0.05 2 0.01 q
Ornithine
0.25 + 0.03
0.21 + 0.01
0.21 + 0.02
0.17 2 0.02
Lysine
0.59 + 0.06
0.64 + 0.05
0.65 k 0.07
0.54 + 0.06
Histidine
0.38 + 0.04
0.40 + 0.03
0.40 k 0.06
0.32 t 0.03
Carnosine
4.90 ‘- 0.66
5.56 2 0.47
5.26 k 0.62
4.82 + 0.34t
Arginine
0.32 2 0.04
0.29 + 0.03
0.37 + 0.04t
0.27 2 0.03(1
Total AA
25.28 2 1.86
27.47 2 2.16
28.08 i 1.88
20.80 ‘- 1.055
Noness AA
19.10 ‘- 0.965
23.20 k 1.71
25.29 2 2.08
25.93 k 1.76
Ess AA
2.08 -c 0.17
2.18 2 0.13
2.15 2 0.16
1.70 t 0.13
BCAA
0.69 + 0.05
0.64 ? 0.04
0.61 + 0.05t
0.50 k 0.04t
Aromatic AA
0.19 + 0.02
0.18 ? 0.01
0.17 + 0.02
0.12 2 o.o1t//
Basic AA
1.38 2 0.21
1.30 k 0.09
1.42 2 0.13
1.08 t- 0.12
NOTE. Values are means k SEM. Abbreviations: AA, amino acids; Ess, essential; BCAA, branched chain amino acids. *Significantly differentfrom
the paired values of day 0 (P < ,051.
tSSignificantly different from the paired values of day 7 (P < .05, P < .Ol, respectively). ll§Significantly different on day 15 as compared with the met-hGH group (P < .05, P < .Ol, respectively). /I#Significantly different alteration between days 7 and 15 as compared with the met-hGH group (P < .05, P < .Ol, respectively).
319
GROWTH HORMONE AND MUSCLE PROTEIN METABOLISM
Table 6. Concentrations
-
of Free Amino Acids in Plasma met-hGH
Day 0
Day 7
Day 15
Controls Day 15
-
63 k 6
69 + 2
66 t 5
Aspartate
Taurine
5+0
520
621
Threonine
158 -+ 8
192 2 lot
192 + 15
17120
Serine
152 _t 9
78 _+ 6
521
135 + 7
168 f 9t
192 2 17
Asparagine
83 2 3
104 t 4*
120 k 18
95 IL 5
Glutamate
26 it 4
32 + 5
40 + 9
25 + 3
Glutamine
694 + 20
761 + 26t
892 + 7211
803 k 35**
Glycine
279 2 17
368 k 20*
387 f 37
387 k 29
Alanine
370 2 25
500 + 40t
706 2 425
511 r 52$*
48 + 3
41 +- 2*
41 -+ 4
313 k 8
247 + 9*
210 -t loll
Citrulline Valine
43 2 4 222 + 1ott
Cystine
21 2 3
20 -t 2
14 -c 2
19 k 2
Methionine
40 _f 3
43 k 3
50 -c 2
34 2 2**
lsoleucine Leucine
99 t 2
88 k 3t
188 ;t 5
164 ‘- 5t
87 + 4
83 2 5
147 t 711
148 2 7
Tyrosine
76 k 4
70 r 3
65 f 21
Phenylalanine
70 + 2
71 _f 2
64 + 35
63 i
Ornithine
67 -+ 7
57 k 3
56 k 4
67 t 7
179 k 91
210 2 12
Lysine
189 r 8
Histidine
87 + 4
Arginine
106 + 10
211 +8 83 + 3 99 + 10
59 + 39 1
74 + 3n
85 i 2#
102 z 16
102 2 85tt
Total AA
3,080 + 97
3,362 t 109”
3,647 t 2390
3,300 +- 114**
Noness AA
1,927 + 72
2,256 r 92t
2,641 + 21 Ill
2,292 f 8855
Ess AA
1,153 +- 30
1,106 t- 30
1,006 2 441
1,008 t 37 453 f 20
BCAA Aromatic
AA
Basic AA
600 + 12
499 + 15$
444 -+ i9n
146 2 6
141 -c4
128 2 48
121 *4
381 + 19
394 k 16
354 t 22
397 t 20
NOTE. Values are means r SEM. *tSSignificantly
different from the paired values of day 0 (P < .05, P < .Ol, P < ,001. respectively).
§ll ISignificantly
different from the paired values of day 7 (P < .05, P < .Ol, P < ,001, respectively).
#‘*Significantly tt$S§§Significantly
different
on day 15 as compared
different
alteration
with the met-hGH group (P < .05, P < ,001, respectively).
from the paired values on day 7 as compared
with the met-hGH
group
(P i
.05, P -. .Ol, P c ,001,
respectively).
influence the free amino acid content in muscle, but after a second week of low nitrogen intake, the levels of several individual amino acids, as well as the total sum of amino acids, declined. This depiction was not seen in the met-hGH group, except for the branched-chain amino acids. Ribosomc analysis is a qualitative estimate of protein synthesis.‘” The technique has been validated in human skeletal muscle under a variety of physiological and clinical situ;,ti ens. ?.‘~ ‘.i”.?7Considerable interindividual differences in the absolute values of ribosome parameters, as well as the magnitude of the changes in these parameters, are seen in connection with protein catabolism. Here, the total concentration of ribosomes decreased during the conditioning period. The decrease of the mean value in the control group was 32%. as compared with 17% in the group later receiving met-hGH treatment. This difference statistically insignificant. When interpretating ribosome analysis, significanr differences between paired samples reflect changes in protein synthesis. It is not advisable to draw conclusions from the magnitude of these changes without great caution.“,‘” When the concentration of polyribosomes was calculated. a reduction was seen after the conditioning not
period. After the treatment period, this reduction persisted in the control group, while the polyribosome content was normalized in the met-hGH group. Short-term starvation depresses muscle protein synthesis in man.‘x,2y Two days of refeeding following 3 days of starvation does not normalize the decrease in ribosome content, but the proportion of polyribosomes is restored.” In the present study, 1 week of met-hGH treatment prevented a decrease in the polyribosome concentration. Among free amino acids of muscle, the decline in the glutamine level is a hallmark of protein catabolism during starvation,16 as in several other muscle protein catabolic states.‘.” Malnutrition states, as represented by patients with anorexia nervosa” or gastric cancer,” show a similar glutamine depletion. After surgical trauma there is a correlation between the decrease in muscle glutamine content and the change in polyribosome concentration,” suggesting a close relationship between glutamine metabolism and muscle protein synthesis.“’ Here, the controls exhibited a decrease in muscle glutamine during the second week of the study, which the met-hGH group did not. On the contrary, plasma glutamine concentration became elevated in both groups, but the increase was most pronounced
320
in the met-hGH group. The increase in the branched-chain amino acids and aromatic amino acids seen after short-term starvation or following surgical trauma was not evident here. On the contrary, a decline of these amino acids in muscle, as well as in plasma, was recorded in muscle irrespective of whether met-hGH was provided or not. Malnutrition is not associated with low levels of branchedchain amino acids. On the other hand, uremic patients, as well as patients with liver insufficiency, often show low values.31 In protein catabolic states, the net flux of free amino acids from the periphery to the splanchnic area increase.” It is not settled whether this transport of amino acids results from a “spill over” in the periphery or from a “suction” in the splanchnic area. It is also unclear what mechanisms regulate such changes in amino acid metabolism and transport. Elevated efflux of amino acids from muscle is accompanied by low levels of free amino acids in the tissue. As described above, such changes are most pronounced for glutamine and other nonessential amino acids, while the essential amino acids stay unaltered or increase in concentration.2.*.14,15~17,28 Here, the normalization of the muscle amino acids in the met-hGH group is indicative of nearnormal amino acid and protein metabolism. This possible effect of met-hGH treatment may be exerted in muscle tissue and/or in the splanchnic area. Obviously, the utilization of amino acids for urea production is low under the influence of met-hGH, but this may be related to the magnitude of the amino acid supply, as well as to other regulative factors. Nitrogen economy is improved when hGH is administered to healthy subjects or patients on a hypocaloric diet.“‘,” Here, an adequate caloric intake was given in combination with an insufficient amount of nitrogen. The first week was a conditioning period for the volunteers to approach a metabolic steady-state and to become comparable during the second week when they were randomized to receive met-hGH or not. Although the characteristics of the volunteers were comparable, some biochemical parameters showed differences during the first week. These dissimilarities, which are likely to be attributable to the comparatively small number of subjects included in each group, were diminished when entering the treatment period during the second week of the study. The delayed influence on muscle amino acids of the nitrogen-deficient diet, and the comparatively slow adjustment of the urinary urea excretion, contrast to the more rapid adaptation seen during total starvation.33 The mechanism of the nitrogen-saving effect of hGH is unclear. The urinary urea excretion is attenuated in association with surgical trauma and burns.“.” This is the case during circumstances where adequate support of both calories and nitrogen is given,‘2213as well as where hypocaloric and/or hyponitrogenous regimens are applied.” During starvation, a more efficient nitrogen utilization is observed in normal-weight,‘0 but not in obese subjects.34 A major part of the hGH effect on whole body nitrogen economy is mediated through urea metabolism.35 In the present work, a diminished urea excretion was accompa-
LUNDEBERG ET AL
nied by a low urea concentration in serum. hGH effects the release of both glucagon and insulin from pancreas,36 and glucagon is the major determinant of urea production3’ Unfortunately, glucagon was not measured in the present investigation, but the augmented levels of insulin and C-peptide were reproduced.“’ No effect on serum glucose in the postabsorptive state was seen. The daily urea excretion declined during the conditioning period to become adapted to an approximately steadystate during the second week (Fig 1). The adaption was quicker in the met-hGH group than in the control group. The differences between the two groups in urea and creatinine excretion are in accord with the literature.10,35 The excretion of ammonia decreased during the second week in the control group. No changes in urinary ammonia are reported in association with hGH treatment.‘0.12X34 In sepsis and after surgical trauma, an increase of the hGH level in serum is seen,35.38which is abolished by spinal anesthesia blocking the response of stress hormones after surgical trauma.3S High hGH levels are associated with elevated levels of corticotropin and corticosteroids,38 but catecholamines do not augment hGH.39 Suppression of hGH sometimes noted in septic subjects is associated with a high mortality.40 This pathophysiological increment of hGH has been suggested to be important for the regulation of protein metabolism.41 Acromegaly in man and pathological overproduction of GH in animals are accompanied by a hypertrophy of skeletal muscle.42 Whole body protein synthesis, as well as whole body protein degradation, is enhanced postoperatively when met-hGH is provided.” However, the role of an unphysiologically high hGH level on human muscle protein metabolism after trauma is not clarified. The results presented here show that a comparatively low dose of met-hGH administered to healthy subjects affects muscle protein metabolism. In addition, the techniques used provided information about the state of muscle protein metabolism. The ribosome technique is an in vivo method that allows repetitive measurements in a patient, to monitor the course of changes in muscle protein synthesis.3*6,43 Studies evaluating the effects of met-hGH on skeletal muscle after elective surgery or in intensive care patients should include several techniques in parallel to assess protein metabolism, resting on different underlying assumptions. To conclude, changes consistent with protein catabolism in skeletal muscle were prevented when met-hGH was provided to healthy subjects on a hyponitrogenous oral diet. Whole body nitrogen economy was improved as shown by a diminished urea excretion and a low serum urea level indicative of a decreased urea production. Investigations of the possible beneficial effect of met-hGH to prevent skeletal muscle vast after surgical trauma are advocated. ACKNOWLEDGMENT We are thankful for the skillful nursing assistance of E. Bostedt and the skillful technical assistance of C. Hebert, L. Thunblad, and Dr M.R. Ah, and to L. Nordhmd for the help in designing computer programs for the amino acid calculations. Somatonorm was generously supplied by KabiVitrum Peptide Hormones, Stockholm, Sweden.
GROWTH HORMONE
AND MUSCLE PROTEIN METABOLISM
321
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