Depression of liver protein synthesis during surgery is prevented by growth hormone

Hans Barle1, Pia Essén1, Björn Nyberg2, Hans Olivecrona2, Michael Tally3, Margaret A. McNurlan4, Jan Wernerman1, and Peter J. Garlick4

1 Department of Anesthesiology and Intensive Care and 2 Department of Surgery, Huddinge University Hospital, S-141 86 Huddinge; 3 Department of Endocrinology, Karolinska Hospital, S-104 01 Stockholm, Sweden; and 4 Department of Surgery, University Medical Center, State University of New York at Stony Brook, Stony Brook, New York 11794-8191


    ABSTRACT
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ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study was undertaken to elucidate the specific effects of growth hormone (GH) on liver protein metabolism in humans during surgery. Otherwise healthy patients scheduled for elective laparoscopic cholecystectomy were randomized into controls (n = 9) or pretreatment with 12 units of GH for 1 day (GH 1, n = 9) or daily for 5 days (GH 5, n = 10). The fractional synthesis rate of liver proteins, as assessed by flooding with [2H5]phenylalanine, was higher in the GH 5 group (22.0 ± 6.9%/day, mean ± SD, P < 0.05) than in the control (16.1 ± 3.1%/day) and GH 1 (16.5 ± 5.5%/day) groups. During surgery, the fraction of polyribosomes in the liver, as assessed by ribosome analysis, decreased in the control group by ~12% (P < 0.01) but did not decrease in the GH-treated groups. In addition, the concentrations of the essential amino acids and aspartate in the liver decreased in response to GH treatment. In conclusion, GH pretreatment decreases hepatic free amino acid concentrations and preserves liver protein synthesis during surgery.

hepatic; laparoscopy; mass spectrometry; stable isotopes


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GROWTH HORMONE (GH) is known to exert a variety of effects on whole body metabolism in healthy humans, including elevated energy expenditure, increased fat oxidation, decreased glucose utilization, insulin resistance, and enhanced whole body protein synthesis (despite hypocaloric nutrition) and nitrogen retention (23, 24, 27). An interesting aspect of GH is its potential for the treatment of catabolic conditions, which has gained increasing attention during recent years. Thus GH reduces net protein loss after surgery of moderate severity (20, 32, 37) and in more severe catabolic conditions, such as sepsis and trauma (10, 36). Furthermore, skeletal muscle protein synthesis is preserved after surgery, when GH is administered (17). However, muscle protein synthesis is depressed by GH treatment in wasted patients with acquired immune deficiency syndrome, even though a stimulation is observed in asympotomatic human immunodeficiency syndrome patients, compared with healthy controls (26).

Most of the knowledge of the influence of GH on liver protein metabolism emanates from studies in animals, which have shown that liver protein synthesis is increased both by GH and a combination of GH and insulin-like growth factor I (IGF-I), but not by IGF-I alone (6, 21, 30). In humans, the effects of GH on plasma concentrations and synthesis rates of hepatic export proteins have been investigated previously, both in healthy subjects and during catabolic states (9, 31).

The aim of this study was to investigate the effects of treatment with GH for 5 h or 5 days on human liver protein metabolism during surgery. The shorter period results in supranormal serum concentrations of GH, while the concentration of IGF-I is maintained, whereas the longer pretreatment results in high serum concentrations of both GH and IGF-I. Two major parameters were investigated, the rate of total liver protein synthesis and the hepatic free amino acid concentrations, in liver biopsy specimens obtained laparoscopically, employing the recently developed procedure for obtaining liver tissue during elective laparoscopic cholecystectomy (1, 3). To facilitate the interpretation of the effect of GH on liver protein synthesis, measurements were made both by the incorporation of [2H5]phenylalanine into liver protein and by the concentration and size distribution of ribosomes in the liver.


    PATIENTS AND METHODS
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ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

L-[2H5]phenylalanine, 99 mole percent (Mass Trace, Sommerville, MA), was dissolved in sterile water together with unlabeled phenylalanine (Ajinomoto, Tokyo, Japan) to a concentration of 20 g/l, 10 mole percent excess (MPE). The solutions were prepared, heat-sterilized, and stored in sterile containers.

Subjects and Preoperative Treatment

Patients (n = 28), scheduled for elective laparoscopic cholecystectomy with no known disease apart from cholecystolithiasis, participated in the study. None of the patients had had any significant weight loss (>2-3 kg) over the last 3 mo before surgery. The patients were randomized into three groups, as follows: nine subjects received no GH treatment and served as controls, nine subjects received one subcutaneous injection of GH (12 units Genotropin; Pharmacia Upjohn, Stockholm, Sweden) 5 h before the operation (GH 1 day), and one group of 10 patients received one subcutaneous injection of GH (Genotropin, 12 U/day) each morning for five preoperative days plus an injection of the same dose the night before the operation (GH 5 days). The three groups were comparable regarding age, weight, and body mass index (Table 1). The nature, purposes, and potential risks of the experimental procedures were explained to the patients before obtaining their voluntary consent. The research protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and had received an a priori approval by the Ethical Committee of the Karolinska Institute (Stockholm, Sweden).

                              
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Table 1.   Characteristics of patients

Experimental Protocol

The patients were studied in the postabsorptive state (after a 12- to 15-h-long fast). Venous blood samples were drawn from all patients for the analysis of concentrations of glucose in blood and of urea, insulin, GH, and IGF-I in serum. This was done in the morning on the day of the operation in control and GH 1 groups of patients and before the GH treatment was initiated in the GH 5 group. In the GH-treated groups, a second blood sample was taken immediately before surgery (see Table 6). Samples for the analysis of urea in serum were taken before surgery only. The basic details of the isotope injection and liver sampling procedure have been described previously (1-3). In short, after premedication, antecubital venous lines were inserted bilaterally, and the patients were given 500-800 ml of Ringer solution (Ringer-Acetat; Pharmacia Upjohn) in the left arm during the procedure. The line in the right arm was used for blood sampling. The anesthesia was induced by giving 5-7 mg/kg sodium thiopentone (Pentothal; Natrium, Abbott Laboratories, Queensborough, Kent, UK), 2.5-5 µg/kg fentanyl (Leptanal; Janssen Pharmaceutica, Beerse, Belgium), and 0.5 mg/kg atracurium (Tracrium; Wellcome Foundation, London, UK). The anesthesia was maintained by giving isoflurane (Forene; Abbott Laboratories) at a concentration corresponding to minimal alveolar concentration 0.75-1.5, measured indirectly as end-tidal values, in a mixture of oxygen-air (30:70), with intermittent doses of fentanyl and atracurium, when needed. After the insufflation of carbon dioxide in the abdomen and the insertion of four trocars through the abdominal wall, the first liver biopsy specimen was taken from the edge of the right liver lobe using laparoscopic scissors for the analysis of the hepatic amino acid concentrations and the concentration and size distribution of ribosomes. This was done 43 ± 14 min after the induction of anesthesia and 20 ± 8 min after the start of the operation, immediately before the surgical procedure, per se (i.e., cholecystectomy), was initiated. At the same time, a blood sample for analysis of plasma amino acid concentrations was taken. Immediately thereafter, the patients received an injection of 45 mg/kg L-[2H5]phenylalanine, 10 MPE intravenously over 10 min in the left arm. A second liver biopsy specimen was taken, 1-2 cm from the first one, 76 ± 15 min after the induction of anesthesia, 51 ± 8 min after the surgical procedure was initiated, and 31 ± 4 min after the injection of phenylalanine, for the analysis of liver protein synthesis and the concentration and size distribution of ribosomes. Blood samples were also drawn at the following intervals: 0, 5, 10, 15, 30, 50, 70, and 90 min after the injection of phenylalanine for the determination of the isotopic enrichment of phenylalanine in plasma. All blood samples were centrifuged at 2,000 rpm for 20 min and were stored in a -80°C freezer pending analysis. All surgical procedures were performed by the same surgeon, and no complications due to bleeding were observed.

Sample Preparation and Analysis of Liver Tissue and Plasma

Free amino acids. The details of the preparation of samples for the analysis of hepatic and plasma amino acid concentrations have been described previously (1). In short, the first liver tissue specimen from each patient was weighed to the nearest 0.1 mg (Cahn 30; Cahn Instruments, Cerritos, CA) and was homogenized in cold 4% sulfosalicylic acid (SSA) containing norleucine as internal standard to precipitate the protein. After centrifugation and adjustment of pH, the free amino acids in the supernatant were analyzed by ion exchange chromatography on a Ultropac 8 Lithium form ion exchange column (202 × 4.6 mm ID, 8 µm particle size; Biochrom, Cambridge, UK) in an automated amino acid analyzer (Alpha Plus; LKB Pharmacia, Bromma, Sweden), using lithium citrate buffers.

Plasma samples for the determination of free amino acid concentrations were treated with 3% SSA to precipitate protein. After centrifugation, ion exchange chromatography was performed, as for liver samples.

Liver protein and plasma enrichment of [2H5]phenylalanine. The details of the preparation and analysis of tissue and plasma samples for the enrichment of L-[2H5]phenylalanine have been described elsewhere (3, 4, 25). Briefly, frozen liver specimens weighing ~20 mg were homogenized in cold 3% perchloric acid (PCA) on ice in Eppendorff microfuge tubes and centrifuged for 5 min at 8,000 rpm at 4°C. The supernatant was saved for the analysis of the enrichment of the free phenylalanine in the liver. The protein precipitate was washed repeatedly to remove traces of free phenylalanine, followed by suspension in 0.3 M NaOH and reprecipitation in 5% PCA. After further washing, proteins were hydrolyzed in 6 M HCl for 24 h at 110°C. The HCl was removed by evaporation in vacuo, and the hydrolyzate was used for the measurement of isotopic enrichment.

Plasma for the determination of free phenylalanine enrichment was treated with 8% SSA to precipitate protein. The amino acid-containing supernatants were purified with cation exchange columns [AG 50, 50W-X8, (H+) form, 100-200 mesh; Bio-Rad, Hercules, CA] eluted with 4 M NH4OH. The samples were then dried in vacuo.

The supernatants for the analysis of the enrichment of the free phenylalanine pool of the liver were neutralized with potassium hydroxide and then centrifuged to remove potassium perchlorate. The samples were acidified again with SSA and prepared in the same way as plasma samples.

Mass spectrometry. Briefly, the enrichment of L-[2H5]phenylalanine from liver protein hydrolyzates was determined by measuring the mass-to-charge ratio (m/z) at 106 and 109 of the n-heptafluorobutyryl derivative of phenylethylamine (prepared by enzymatic decarboxylation) on a Fisons MD 800 mass spectrometer (Fisons, Beverly, MA) under electron ionization. With this instrument, there was no change in the enrichment with sample loading, and the previously employed calibration curves were not necessary (4). The L-[2H5] enrichment of free phenylalanine in plasma and liver was measured by monitoring the ions at m/z 336 and 341 of the t-butyldimethylsilyl derivative on a HP 5972 mass spectrometer (Hewlett Packard, Palo Alto, CA) under electron ionization.

Ribosome analysis. The method for assessing tissue protein synthesis by analyzing the concentration and size distribution of ribosomes has been used extensively in animal studies, e.g., Ref. 19, and it was later applied to investigate human skeletal muscle protein synthesis (38). In short, the two liver biopsy specimens from each patient were weighed and frozen in liquid nitrogen within 1 min. Subsequently, samples were homogenized in a medium containing ribonuclease inhibitor and then were centrifuged at low speed (1,500 g for 10 min). The supernatant was ultracentrifuged for 2 h at 102,000 g (Beckman Instruments, Palo Alto, CA). The ribosome pellet obtained was resuspended and layered onto a density gradient of 0.4 and 1.5 M sucrose. After ultracentrifugation for 60 min (149,000 g; Beckman Instruments), the gradient was pumped through a continuous-flow cuvette, and the absorbance was registered at 260 nm. The area under the curve was measured, and the percentage proportion of polyribosomes in the total ribosome area was calculated. Another part of the ribosome suspension was used to determine the total ribosome concentration by the absorbance at 260 nm and was expressed as optical density per milligram wet weight tissue.

Other Analytical Procedures

Blood glucose and serum urea were analyzed with routine clinical laboratory methods. RIAs were used to determine the plasma concentrations of insulin, GH, and IGF-I (15, 22). Samples for the determination of IGF-I were acid-ethanol extracted and cryoprecipitated, before the RIA step. To eliminate the action of IGF-binding proteins, des-(1---3)-IGF-I was used as a ligand.

Calculations and Statistics

The fractional synthesis rate (FSR) of liver protein, i.e., the total rate of protein synthesis expressed as a fraction of the liver protein, was calculated according to the previously described formula (13)
FSR = P × 100/AUC
where FSR is expressed as percent per day, P is the isotopic enrichment of phenylalanine in liver protein at the time of the biopsy specimen (MPE), and AUC is the area under the curve for plasma free phenylalanine enrichment (MPE) versus time.

Data are presented as means ± SD. Amino acid concentrations, FSR of total liver protein, ribosome analyses, and blood chemistry in the three groups were compared using the Duncan's multiple range test, whereas Student's t-test was used for comparison within the groups.


    RESULTS
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ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The fractional rate of protein synthesis in the liver was 22.0 ± 6.9%/day in the GH 5 group compared with 16.1 ± 3.1%/day in the control group (P < 0.05). The corresponding value in the GH 1 group was 16.5 ± 5.5%/day, which was significantly different from the GH 5 group (P < 0.05, Table 2).

                              
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Table 2.   Fractional synthesis rates of total liver protein

To evaluate the extent that the plasma free phenylalanine enrichment reflected that in tissue, the ratio of enrichments in the liver/plasma at the time of the second liver biopsy specimen was calculated for each patient. The result was 0.96 ± 0.05 in the control group, 0.98 ± 0.06 in the GH 1 group, and 0.98 ± 0.03 in the GH 5 group, thus verifying that equilization of enrichment in the different free amino acid pools ("flooding") had occurred in all groups.

The polyribosomes, as a fraction of total ribosomes, were determined in each of the two liver biopsy specimens in patients from whom sufficient liver tissue was obtained (Table 3 and Fig. 1). The proportion of polyribosomes decreased significantly in the course of surgery in the control group (P < 0.01), whereas this fraction remained unchanged in the other two groups. The change during the course of surgery differed significantly among groups. The change seen in the GH 5 group (+2.2 ± 3.6%, n = 8) was not different from that of the GH 1 group (+1.4 ± 4.4%, n = 6). The corresponding value in the control group was -6.6 ± 3.9% (n = 6), which was significantly different from that of the GH 5 group (P < 0.01) and from the GH 1 group (P < 0.01). Furthermore, the proportion of polyribosomes in the second liver biopsy specimen was lower in the control group compared with both GH-treated groups (P < 0.05 in both cases). The results of liver protein synthesis as assessed by the flooding technique in these three subset groups of patients in which ribosome analysis was performed were similar to the results stated above.

                              
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Table 3.   Concentration and size distribution of ribosomes



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Fig. 1.   Proportion of polyribosomes in two consecutive liver biopsy specimens in each patient, taken at a 30-min interval ("0" and "30" min). GH 1, growth hormone (GH) administered for 1 day; GH 5, GH administered daily for 5 days. A significant fall in the proportion of polyribosomes between the first and second liver biopsy specimen was observed in the control group (* P < 0.01). Furthermore, a difference in the change of the fraction of polyribosomes between the two biopsy specimens between controls and the GH 5 group was observed (*P < 0.01). open circle , Individual patients; , mean values.

The most marked changes in hepatic amino acid concentrations were seen in the GH 5 group compared with the control group (Table 4). Significant decreases in concentration were observed with aspartate, asparagine, valine, methionine, leucine, lysine, and histidine, as well as for the basic, the branched-chain, and the essential groups of amino acids. In the GH 1 group of patients, a similar pattern was observed, with significant decreases for threonine, asparagine, methionine, leucine, lysine, and histidine, as well as for the basic, the branched-chain, and the essential groups of amino acids.

                              
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Table 4.   Hepatic amino acid concentrations

In the GH 5 group, plasma amino acids showed a similar, but less pronounced, pattern of changes with lower concentrations of the basic, the branched-chained, and the essential amino acids compared with controls, whereas no differences were seen when GH 1 patients were compared with controls (Table 5).

                              
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Table 5.   Plasma amino acid concentrations

In the basal state, the concentrations of blood glucose and serum insulin, GH, and IGF-I were similar in the three groups (Table 6). A 10-fold increase of GH in serum (P < 0.01) was observed 5 h after GH administration, whereas the other parameters remained unchanged. Pretreatment with GH for 5 days increased the serum concentration of GH sixfold (P < 0.001) and the concentration of IGF-I threefold (P < 0.001). In parallel, the serum concentration of insulin increased about three times (P < 0.001), whereas the blood concentration of glucose increased ~20% (P < 0.001; Table 6). Immediately before surgery, the serum concentration of urea was lower in the GH 5 group compared with either the control group or the GH 1 group (P < 0.05; Table 6).

                              
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Table 6.   Blood glucose and serum hormone and urea concentrations


    DISCUSSION
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ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Liver protein synthesis, measured during laparoscopic surgery, was higher after a 5-day treatment with GH than in controls (Table 2). However, based on the evidence from the simultaneous ribosome analysis, showing a decrease in polysome concentration in the control group during surgery, as well as previous results, the conclusion that GH induces a direct stimulation of liver protein synthesis may be too simplistic. Instead, GH given for 5 days may counteract a decrease in liver protein synthesis elicited by the surgical procedure. In contrast, a single injection of GH, given 5 h before the liver biopsy specimen (GH 1), did not influence liver protein synthesis compared with controls (Table 2). Furthermore, the free amino acid concentrations in liver and plasma decreased in response to GH treatment.

Human liver protein synthesis has previously been assessed by the incorporation of labeled phenylalanine in liver protein (2, 3). In the present study, this technique was employed to further evaluate the effect of GH on liver protein metabolism. In addition, a study of liver ribosomes was included in the investigation, to facilitate the interpretation of the results of the stable isotope technique analysis. Thus, in patients where sufficient biopsy material was available, the two liver biopsy specimens taken at different time points during the laparoscopic procedure were analyzed for the concentration and size distribution of ribosomes. The total ribosome concentration represents the total capacity for protein synthesis, whereas the percentage of polyribosomes reflects the proportion of ribosomes actively involved in protein synthesis (38). This method is qualitative and therefore has its greatest applicability when repeated measurements in the same subjects are possible. In the control group, a significant decrease in the fraction of polyribosomes was observed between the two biopsy specimens taken ~30 min apart during surgery (P < 0.01, Table 3 and Fig. 1). These data suggest a decline in liver protein synthesis, induced by the surgical procedure. Such a decline is consistent with results of liver protein synthesis rates as assessed by [2H5]phenylalanine in the control group of this study compared with the results of the two previous studies illustrated in Fig. 2. In the first of these studies (3), liver protein synthesis was measured early during the laparoscopic procedure, i.e., after ~18 min of surgery, and the FSR of liver protein was 24.7 ± 3.1%/day (n = 9). However, in the second study (2), where measurements were performed after ~45 min of surgery, liver FSR was much lower, 17.7 ± 3.8%/day (n = 9). Moreover, the latter result is comparable to the value in the control group of the present study, 16.1 ± 3.1%/day, where the measurement was also performed well into the course of the procedure, i.e., after ~52 min of surgery (Table 2 and Fig. 2). Thus two completely different methods for assessing liver protein synthesis have produced results that are consistent in suggesting that the metabolic stress associated with laparoscopic surgery has an almost immediate depressive effect on liver protein synthesis.


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Fig. 2.   Relation between the length of surgery and the fractional synthesis rate of total liver protein as assessed by the flooding technique in three different studies. Values are shown as means ± SD. , data from Barle et al., n = 9 patients (3); black-diamond , data from Barle et al., n = 9 (2); , data from the control group of the present study, n = 9.

After pretreatment with GH for 5 days, there was no difference from the control group, neither in total ribosome concentration nor in the fraction of polyribosomes of the first biopsy, suggesting that GH had no long-term effect (Table 3). However, there was also no decrease in the fraction of polyribosomes between the two biopsy specimens taken ~30 min apart during surgery (Table 3 and Fig. 1). This led to a difference in the proportion of polyribosomes at the time of the second biopsy specimen between controls and the GH 5 group (Table 3). Furthermore, the decrease in the fraction of polyribosomes over time in the control group was significantly different from the change in the GH 5 group (P < 0.01, Fig. 1). Thus, compared with the control group, no decline in liver protein synthesis during surgery was observed in the GH 5 group when assessed with ribosome analysis. This result is consistent with the higher value for liver FSR found in the GH 5 group, 22.0 ± 6.9%/day (Table 2). Although measured after ~50 min of surgery, this value is similar to that obtained after only 18 min of surgery in our earlier study mentioned above (24.7 ± 3.1%/day; see Ref. 3). These results support the hypothesis that pretreatment with GH for 5 days counteracts the decrease in liver protein synthesis that occurs during the laparoscopic procedure.

In the GH 1 group, the measurements of liver protein synthesis were intermediate between the other two groups. However, the FSR in the GH 1 group was close to the value for the control group, whereas results of polyribosome analysis showed a change in the GH 1 group that was close to that of the GH 5 group (Tables 2 and 3, Fig. 1). The reason for this is not clear, but it could be that the values are intermediate between the extremes of the control and GH 5 groups, so that chance determines whether a measurement for the GH 1 group comes nearer to one extreme or the other. However, it does seem that GH given 5 h before surgery is insufficient to prevent fully the decline in liver protein synthesis observed during surgery.

The hepatic amino acid concentrations were measured in tissue samples from the first liver biopsy specimen, when the effect of the surgical trauma was likely to be negligible. Five hours after a single injection of GH, decreased hepatic free concentrations of the essential amino acids were observed. These changes were further augmented after 5 days of pretreatment with GH, when also a significant decrease of the concentration of aspartate was observed (Table 4). The significance of aspartate in the liver is highlighted by its importance as an intermediary in several metabolic pathways that are involved in the degradation of amino acids (e.g., transamination and the urea cycle), and thus the decrease may possibly reflect the anabolic actions of GH. Given the findings that liver protein synthesis rates were higher, and serum urea concentrations were lower (by ~42%), after GH treatment for 5 days compared with controls, this could imply that the hepatic free amino acids were being preferably utilized for protein synthesis instead of oxidation (Tables 2 and 6). However, in the GH 1 group, liver FSR and serum urea concentrations were unaltered compared with controls, despite the changes in the hepatic amino acid concentrations. It is possible that the time period of 5 h after a single injection of GH was too short to be reflected in liver FSR or serum urea concentrations. However, other mechanisms may be involved also. For example, it is possible that the decreased hepatic amino acid concentrations in response to GH treatment were due to a retention of substrate in the periphery. Previous results show that the effect of short-term administration of GH (for 2 days) seems to be exerted exclusively through effects on amino-to-nitrogen conversion in the peripheral tissues (39). However, after prolonged treatment (for 10 days), GH acts directly on the liver and is associated with an absolute decrease in ureagenesis (40). Furthermore, GH may also decrease the hepatic amino acid uptake (29). The similarity of the changes of the hepatic amino acid concentrations after 5 h or 5 days suggests an effect elicited by GH directly. However, the plasma amino acid concentrations were unaltered in the GH 1 group, whereas a decrease in the essential amino acids was seen in the GH 5 group (Table 5). As the plasma amino acid concentrations were unaltered despite the changes seen in the liver free amino acids after 5 h, it is likely that tissues other than the liver contribute to the changes in plasma seen after 5 days of treatment.

The serum concentration of IGF-I in the GH 5 group increased significantly as a result of the treatment with GH for 5 days (Table 6). The effects of GH on whole body protein metabolism are in part mediated by IGF-I, which also has a nitrogen-sparing effect in itself (7). Furthermore, the effects of IGF-I are modified by its binding proteins (16). However, in spite of (or because of) the liver being the major production site for IGF-I (16), virtually no receptors for IGF-I in the adult human liver have been identified so far (5). Even though a more recent study suggests the presence of significant levels of mRNA for receptors of IGF-I in the adult human liver (33), it is most likely that the effect of GH treatment on liver protein and amino acid metabolism should be attributed to GH alone.

When considering the possible anabolic effects of GH on liver protein metabolism, the potential importance of insulin should also be taken into consideration, since the plasma insulin levels were elevated in the GH 5 group (Table 6). Even though insulin does not induce an increase in lean body mass, its presence may be a prerequisite to enable the anabolic actions of GH (34). However, early in vitro studies have shown that insulin (and amino acids) acts by suppressing protein degradation rather than by stimulating protein synthesis in the liver (28, 41). In our previous study, where the patients were given short-term parenteral nutrition, plasma insulin concentrations increased fivefold compared with controls (2), but no effect on liver protein synthesis rates was observed. Moreover, insulin has no effect on protein synthesis in human skeletal muscle (25).

Liver tissue sampling during laparoscopic surgery offers an alternative to traditional techniques to obtain liver tissue for scientific purposes, providing a possibility to acquire the liver biopsy specimens in a safe and reproducible manner from healthy subjects. However, as shown in this study, the exact time point for liver tissue sampling during the course of surgery should be chosen with care since the anesthesia and/or the surgical procedure may rapidly affect liver protein metabolism. Thus it is possible that early sampling reflects relatively normal physiological conditions, whereas sampling performed later during the course of the procedure may reflect the effect of surgical stress (Fig. 2). This effect is similar to that in muscle tissue, where uncomplicated surgery in the form of open cholecystectomy, but not general anesthesia in itself, induces an immediate (i.e., within 90 min after surgery) decrease of muscle protein synthesis by 30% (11). Although laparoscopic cholecystectomy has been shown to have less pronounced metabolic effects compared with open cholecystectomy, including a smaller increase of urea synthesis (14) and a smaller reduction of insulin sensitivity (35), the same decrease in muscle protein synthesis is observed after 24 h, irrespective of the surgical approach (12).

A number of studies, primarily in animals, show that liver protein synthesis rates increase prominently during extreme states of catabolism (major trauma/sepsis), which probably also applies in humans (8, 18). However, the immediate effect of surgical trauma on liver protein synthesis in humans has not been characterized before. Taken together, the results from this and previous studies indicate that the effect of a catabolic insult on liver protein synthesis may be graded and that the effect is dependent on the time factor (i.e., when in relation to the insult, protein synthesis is measured) as well as the size of the trauma and/or the severity of the systemic inflammation. This implies that if liver protein synthesis rates would have been measured later after the surgical procedure (which would be difficult from a practical point of view) and/or after a trauma of greater magnitude, the results may well have been different. This issue needs to be investigated further in humans.

In conclusion, by combining a stable isotope procedure with analysis of polyribosomes, it has been possible to detect a fall in the rate of liver protein synthesis during the course of laparoscopic cholecystectomy. The effect can be overcome by pretreatment of the patient with GH for 5 days, but a single treatment 5 h before surgery was not effective. This protective effect of GH does not involve a change in the basal rate of hepatic protein synthesis but may provide further insight into the anticatabolic role of this hormone.


    ACKNOWLEDGEMENTS

The skilled technical and nursing assistance of George Casella, Viveka Gustavsson, Christina Hebert, and Eva Skoog is gratefully acknowledged.


    FOOTNOTES

This investigation was supported by grants from the Swedish Medical Research Council (Project 04210), the Maud and Birger Gustavsson Foundation, and the County Council of Stockholm. H. Barle is the grateful recipient of the 1993 European Society of Parenteral and Enteral Nutrition-Clintec scholarship.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for correspondence and reprint requests: H. Barle, Dept. of Anesthesiology and Intensive Care, K 32, Huddinge Univ. Hospital, S-141 86 Huddinge, Sweden (E-mail: barle{at}ebox.tninet.se).

Received 14 August 1998; accepted in final form 4 December 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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2.   Barle, H., B. Nyberg, K. Andersson, P. Essén, M. A. McNurlan, J. Wernerman, and P. J. Garlick. The effects of short-term parenteral nutrition on human liver protein and amino acid metabolism during laparoscopic surgery. JPEN J. Parenter. Enteral Nutr. 21: 330-335, 1997[Abstract].

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4.   Calder, A. G., S. E. Anderson, I. Grant, M. A. McNurlan, and P. J. Garlick. The determination of low d5-phenylalanine enrichment (0.002-0.09 atom percent excess), after conversion to phenylethylamine, in relation to protein turnover studies by gas chromatography/mass spectrometry. Rapid Commun. Mass Spectrom. 6: 421-424, 1992[Medline].

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