Growth Hormone Regulates Amino in Human and Rat Liver

Acid Transport

ANTHONY J. PACITTI, M.S., YOSHIFUMI INOUE, M.D., DONALD A. PLUMLEY, M.D., EDWARD M. COPELAND, M.D., F.A.C.S., and WILEY W. SOUBA, M.D., Sc.D., F.A.C.S.

Human growth hormone (GH) has been shown to improve nitrogen balance in surgical patients and to decrease urea production. This has been thought to be due primarily to an increase in protein synthesis in skeletal muscle. Little attention has focused on the liver as a possible site where GH may modulate amino acid uptake and thereby divert nitrogen away from ureagenesis. The authors hypothesized that GH regulates amino acid transport in hepatocytes at the plasma membrane level. They studied hepatic amino acid transport in 20 healthy surgical patients that received saline, low-dose GH (0.1 mg/kg/day), or high-dose (0.2 mg/kg/day) GH for 3 days before operation. At operation, a 5- to 10-g wedge biopsy of the liver was obtained, and hepatocyte plasma membrane vesicles were prepared by Percoll density gradient centrifugation. Vesicle transport of [3HJMeAIB, a highly selective system A substrate, and [3H]-glutamine, a selective system N substrate, was measured, employing a rapid mixing/filtration technique. Hepatocyte plasma membrane vesicles were also prepared from 14 rats treated with saline or one of three different GH treatment regimens: (A) 12 hours after chronic GH treatment (6 mg/kg every 12 hours X 4 doses); (B) 4 hours after acute (1 dose) GH treatment; and (C) 4 hours after chronic GH treatment. In human liver vesicles, low-dose GH resulted in a 13% decrease in system A activity (p = not significant), whereas high-dose GH caused a marked 79% decrease (6.7 ± 1.7 pmol/mg protein/10 seconds in control patients versus 1.4 ± 0.7 in GH, p < 0.05). System N was unaffected. Kinetic analysis of MeAIB transport by vesicles from high-dose GH patients showed the reduction in transport to be due to a 63% decrease in the V.,, (maximal transport velocity) with no alteration in the transport Km (carrier affinity). Vesicles from rats treated chronically with GH using a protocol similar to that used for human subjects exhibited decreased system A transport activity (10.4 ± 0.4 pmol/mg pro/10 seconds in controls versus 7.5 ± 0.2 in GH, p < 0.05) secondary to a 59% reduction in the transport V,,,,. Chronic growth hormone treatment decreases the activity of system A in both human and rat hepatocytes. This Presented at the I 12th Meeting of the American Surgical Association, April 6-8, 1992, Palm Desert, California. Supported by NIH grant CA 45327 and a grant from the GI Study Section of the Veterans Administration Merit Review Board (Dr. Souba). Address reprint requests to Wiley W. Souba, M.D., Sc.D., Department of Surgery, Box J-286, JHMHSC, Gainesville, FL 326 10. Accepted for publication April 10, 1992.

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From the Department of Surgery, University of Florida College of Medicine, Gainesville, Florida

may be one mechanism by which GH diminishes hepatic ureagenesis and spares amino acids for peripheral protein synthesis.

E a ROSION OF LEAN body mass and negative nitrogen balance are characteristic of catabolic insults such

as major injury and infection.'2 When the disease process is prolonged, the patient may become sufficiently malnourished such that wound healing is impaired, susceptibility to infection is increased, and muscle strength is diminished.2'3 Although the initial enthusiasm for providing specialized nutrition to critically ill surgical patients was great, several reports in the past decade indicate that aggressive nutritional support does not prevent negative balance and erosion of lean body mass during severe catabolic illness.4 Consequently, more recent attention has focused on alternative methods of modifying or attenuating the catabolic response to injury and infection.5 Human growth hormone (GH) is a single-chain polypeptide of 191 amino acids. It is the most abundant hormone in the pituitary gland, and its growth-promoting properties are well known. The anabolic effect of growth hormone administration is associated with nitrogen retention and is reflected by a diminished urea excretion that results from a true reduction in urea production in the liver.3'6'7 The mechanisms underlying the shift of nitrogen from ureagenesis into cell growth have not been fully elucidated, however. Although the acute effects of growth hormone include stimulation of the system A membrane amino acid transporter,8-10 the long-term effects of multiple doses of growth hormone are unclear. Welbourne et al.7 demonstrated that GH administration to hypophysectomized rats resulted in a redistribution of glutamine nitrogen into glutamate and away from urea; simultaneously there was a 30% decrease in hepatic ala-

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nine extraction in GH-treated animals in the face of unchanged circulating alanine levels. These observations are consistent with a decrease in the hepatic transport of alanine, a key ureagenic amino acid. In the current investigation, we have examined the effects of chronic growth hormone administration on hepatocyte plasma membrane amino acid transport activity, focusing on system A and system N, the two agencies known to mediate the transmembrane flux of alanine and glutamine, respectively. We hypothesized that alterations in the intrinsic activity of these putative hepatic carrier proteins underlies a potential mechanism at the level of the plasma membrane whereby the liver contributes to the increase in nitrogen retention secondary to growth hormone administration. Materials and Methods Studies were carried out using human subjects, and parallel rat studies also were performed. Twenty healthy adult male surgical patients (32 to 68 years of age) requiring elective abdominal surgery at the University of Florida Shands Hospital or the Gainesville Veterans Administration Hospital were studied. Entry criteria for participation in the study protocol included (1) no evidence of organ dysfunction by preoperative assessment (i.e., patients with liver dysfunction, renal insufficiency, diabetes, or lung disease were excluded), (2) no conclusive evidence of or history of cancer, (3) the need for elective abdominal surgery, (4) no history of recent weight loss, and (5) willingness to participate in the study by giving informed consent. The operations performed included cholecystectomy, Nissen fundoplication, right hemicolectomy, vagotomy and pyloroplasty, liver resection, and pancreaticojejunostomy. All patients were consuming a regular diet up to the day before surgery and were made NPO (nothing by mouth) for 12 to 24 hours before the operation. Patients were randomized to receive human methionyl recombinant growth hormone (hrGH, Protropin, Genentech, Inc., San Francisco, CA) in a dose of 0.1 mg/kg (low-dose GH, n = 6), or 0.2 mg/kg hrGH (high-dose GH, n = 8) each day for 3 days before operation. Control patients (n = 6) did not receive GH. Growth hormone was reconstituted in sterile water and was administered subcutaneously in the thigh each evening at 8:00 P.M. for three consecutive evenings before the morning of surgery. At operation, a 5- to 10-g wedge biopsy of the liver was obtained on entering the abdominal cavity before proceeding with the formal operative procedure. The biopsy was used to prepare membrane vesicles for amino acid transport studies. Gross or histologic evidence of cirrhosis precluded inclusion of the sample in the transport assays. The study was approved by the Institutional Review Board of the Shands Hospital and the University of Florida and

Ann. Surg. * September 1992

by the Subcommittee for Clinical Investigation at the VA Hospital. Informed consent was obtained from each patient by one of the authors (WWS or DAP). Fourteen adult male Sprague-Dawley rats weighing 200 to 230 g were obtained from Harlan Industries (Indianapolis, IN) and used for parallel rat studies. Animals were housed in the animal care facility, exposed to 12hour light-dark cycles, and given access to standard rat chow and water ad libitum. Rats were treated with growth hormone using three separate protocols. In the first of these, rats received multiple injections of GH (6 mg/kg in sterile water intraperitoneally every 12 hours X four injections) and vesicles prepared 12 hours after the last GH injection (Chronic GH- 12 hour). In the second group, rats received a single intraperitoneal injection of GH (6 mg/kg), and vesicles were prepared 4 hours later (Acute GH-4 hour). The third group received the same GH treatment as the first group (6 mg/kg intraperitoneally every 12 hours X 4 injections) and vesicles were prepared 4 hours after the last injection (Chronic GH-4 hour). Control animals received intraperitoneal injections of sterile water. All animals were fasted overnight before being killed. Animal experiments were approved by the Committee for the Use and Care of Laboratory Animals at the University of Florida. Preparation ofHepatic Plasma Membrane Vesicles Hepatic plasma membrane vesicles (HPMVs) from human subjects were prepared using a modification of the method described by Prpic et al. " At surgery, a 5-g wedge biopsy of liver was obtained, placed on ice, and immediately taken to the laboratory to begin processing for vesicles without prior freezing. All procedures were performed at ice-cold temperatures. Liver samples were perfused using a syringe with approximately 50 mL ice cold PBS (phosphate-buffered saline; 150 mmol/L NaCl, 10 mmol/L Na2HPO4, pH 7.4) until blanched free of gross blood. Samples then were minced with scissors in approximately 35 mL SEB (sucrose-EGTA [ethylene glycol tetra-acetic acid] buffer; 250 mmol/L sucrose, 1 mmol/L EGTA, 10 mmol/L HEPES, pH 7.5) and homogenized for 20 seconds on setting #6 using a Polytron (Kinematica, Switzerland, Brinkman Instruments, Westbury, NY), followed by an additional 15-second homogenization. The homogenate was diluted to 6% (wt/vol) with SEB and centrifuged at 1 50g for 2 minutes to remove gross particulate matter. The supernatant was then centrifuged at 1464g for 10 minutes and the resultant pellets were pooled, brought to approximately 60 mL with SEB, and resuspended with a Dounce homogenizer by 10 passes of a loose-fitting pestle followed by four passes with a tightfitting pestle. The suspension was filtered through 8-ply gauze, added to 13.7 mL Percoll (Pharmacia, Piscataway,

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NJ), and brought to a final volume of 115 mL with SEB (1 1.9% vol/vol final [Percoll]). The Percoll suspension was thoroughly mixed, transferred to three 50-mL clear polycarbonate test tubes, and centrifuged at 34,540g for 30 minutes. The resultant plasma membrane bands were harvested with a 3-cc syringe, pooled, and diluted 1:6 (vol/ vol) with SMB (sucrose-magnesium buffer; 250 mmol/L sucrose, 1 mmol/L MgCl2, 10 mmol/L HEPES, pH 7.5). The plasma membrane suspension was centrifuged at 34,540g for 30 minutes and the membrane pellets resuspended and vesiculated in SMB by three passes through a 22-gauge needle to an approximate concentration of 3 to 6 mg membrane protein/mL. Vesicles were aliquoted into Nunc cryotubes (InterMed, Denmark) and stored at -70 C until studied. For studies employing rat liver, vesicles were prepared essentially as described above with minor modifications. The entire rat liver (7 to 9 g wet weight) was used to prepare vesicles, and a single homogenization step using the Dounce hand-held homogenizer was employed before initial centrifugation in place of the dual Polytron and Dounce homogenization employed for human liver. Vesicle yield ranged from 1.5 to 3.0 and 3.0 to 5.0 mg membrane protein/g wet weight liver for human and rat liver, respectively. Hepatic plasma membrane vesicle purity and integrity of selected samples was routinely evaluated in our laboratory by determining the relative enrichment of the plasma membrane enzyme marker 5'nucleotidase'2 and the microsomal enzyme markers glucose-6-phosphatase,'3 and nicotinamide-adenine dinucleotide phosphate cytochrome c reductase,'4 and by evaluation of membrane transport and permeability characteristics. Inorganic phosphate was determined according to the method of Fiske and Subbarow.'5

30 ,L Na+- or K+-uptake buffer in 12 X 75-mm polystyrene tubes using an electronic timer/vortexer apparatus. At various times, the uptake ofamino acid was terminated by the addition of 1 mL ice-cold PBS. The mixture was immediately passed over a 0.45-,um nitrocellulose filter under low-pressure vacuum filtration. The filter was rapidly washed twice with 4 mL ice-cold PBS/wash. Filters then were dissolved in 10 mL Aquasol (New England Nuclear, Boston, MA) and trapped radioactivity determined in a liquid scintillation counter (Beckman LS 7800, Beckman Scientific Instruments, Irvine, CA) Sodium-dependent transport was determined by subtracting uptake in the presence of potassium (Na+-independent uptake, triplicate or quadruplicate samples) from that observed in the presence of sodium (total uptake, quadruplicate samples). All transport assays were carried out at 22 C. To evaluate transport kinetics, assays were undertaken as described above with the final initial extravesicular amino acid concentration varied from 50 ,mol/L to 10 mmol/ L. Osmotic adjustments for the varying concentrations of amino acid were made with sucrose. Kinetic experiments were performed at the 10- or 20-second time point under initial rate conditions. Blank values (no vesicles present) were subtracted from uptake values in all transport experiments. Protein determinations were performed by a modified Lowry method.'6 All data were normalized to membrane protein and are expressed as Na+-dependent transport in pmol/mg protein/unit time.

Measurement of Amino Acid Transport by Membrane Vesicles

Results Three patients were excluded from the study because of the presence of mild cirrhosis (one patient) and because of the inability to obtain a biopsy secondary to adhesions (two patients). Preparation of hepatic plasma membrane vesicles resulted in routinely consistent 12- to 15-fold enrichments in the specific activity of the plasma membrane enzyme marker 5'-nucleotidase and 25% to 35% impoverishments in the microsomal enzyme markers glucose-6-phosphatase and NADPH:cytochrome c reductase relative to the activity observed in the initial crude liver homogenates. Vesicular transport of amino acids was stimulated by Na+ and vesicular distribution ratios of 1.5 to 2 and 4 to 5 were attainable in the presence of Na+ for MeAIB and glutamine, respectively. These data are consistent with membrane vesicles of plasma membrane origin and functional integrity.

Human or rat hepatic plasma membrane vesicles (HPMVs) were thawed at room temperature and resuspended by three passes through a 22-gauge needle/1-cc syringe. Vesicles were diluted to a protein concentration of 2.5 to 3.5 mg/mL with SMB and kept on ice. Amino acid transport activity was determined in both the presence and absence of sodium. Final assay concentrations in the Na+-uptake buffer were 100 mmol/L NaCl, 1 mmol/L MgC92, 10 mmol/L HEPES, pH 7.5, and varying concentrations, depending on the experiment, of L-[3H]glutamine (Amersham, Arlington Heights, IL) or [3H]methylaminoisobutyric acid (MeAIB; American Radiolabeled Chemicals, Inc., St. Louis, MO). The K+-uptake buffer was identical, with the exception that NaCl was replaced by 100 mmol/L KCI. Transport was initiated by mixing 30 ,uL vesicles (75-100 ,ug membrane protein) with

Statistical Analysis Data were analyzed by either analysis of variance followed by Fisher least squared difference test if a significant F-value was obtained or Student's t test where appropriate. The level of significance was set at 0.05.

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The 10-second Na+-dependent transport of MeAIB and glutamine by HPMVs from control and GH-treated patients is shown in Figure 1. Low-dose GH treatment resulted in a trend toward a 13% reduction in MeAIB transport, whereas high-dose treatment resulted in a significant 79% decrease in the transport of 100 ,umol/L MeAIB (Fig. 1A; p < 0.05). Conversely, GH treatment had no effect on the transport of 100 jumol/L glutamine by HPMVs prepared from human liver (Fig. 1 B). To investigate the nature of the decrease in the transport of MeAIB by HPMVs from patients treated with highdose GH, kinetic studies were undertaken. The Na+-dependent initial rate of MeAIB transport by vesicles from

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FIG. 1. Amino acid transport activity in HPMVs from control and growth hormone-treated patients. Patients received saline (control), 0.1 mg/kg (low-dose GH), or 0.2 mg/kg (high-dose GH) for 3 days before operation. At surgery, liver biopsy specimens were obtained and membrane vesicles prepared. The Na+-dependent transport of(A) 100 ,mol/L [3H]-MeAIB or (B) 100 ,umol/L [3H]-glutamine by vesicles was determined. Data are mean ± SEM. * p < 0.05 by ANOVA.

control and high-dose GH-treated patients was determined across increasing extravesicular MeAIB concentrations from 50 ,umol/L to 10 mmol/L. Representative data are shown in Figure 2. The initial MeAIB transport rate by vesicles from patients treated with high-dose GH was markedly attenuated across all MeAIB concentrations relative to that of vesicles from control patients (Fig. 2A). Eadie-Hofstee linear transformation of the data demonstrated that the reduction in transport activity was secondary to a 63% decrease in the maximal velocity of

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transport with no alteration in transporter affinity

(Fig. 2B). Previous studies have established that Na+-dependent glutamine transport in the rat occurs almost exclusively by system N.'7 The situation in human liver is more equivocal, however, with some suggestion that a significant portion of glutamine transport occurs through system A. 8 Accordingly, we examined the Na+-dependent transport of 100 umol/L glutamine by human HPMVs in the presence of a 10 mmol/L excess of unlabeled MeAIB, to block any contribution of system A to glutamine uptake. As shown in Figure 3, the presence of MeAIB had no effect on the transport of glutamine by HPMVs from human subjects. The lack of efficacy of MeAIB in inhibiting glutamine transport was equally evident in all three patient groups and, therefore, the data from all patients was pooled to illustrate the lack of an effect of MeAIB. Rat Studies Parallel studies were undertaken to examine the effect of GH treatment on hepatic amino acid transport using three different protocols. In the first of these, rats were treated with 6 mg/kg GH every 12 hours X 4 doses, and HPMVs were prepared 12 hours after the last GH dose. This protocol was chosen because of its similarity to that used in the human studies, that is, chronic GH treatment and approximately a 12-hour lag period from the last exposure to GH and harvesting of liver. Qualitatively similar to the data obtained for human liver, prior treatment of animals with GH significantly attenuated the vesicular transport of 50 rmol/L MeAIB by 28% (Fig. 4; p < 0.05).

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FIG. 4. Amino acid transport activity in HPMVs from control and growth hormone-treated rats. Rats received 6 mg/kg GH every 12 hours X 4 doses and hepatic plasma membrane vesicles were prepared 12 hours after the last injection of GH. The Na'-dependent transport of 50 Mmol/ L [3H]-MeAIB or 100 Asmol/L [3H]-glutamine by vesicles was determined. Data are mean ± SEM. * p < 0.05.

Although glutamine transport exhibited a trend toward increase in activity, this did not achieve statistical significance. A representative time course of the Na+-dependent transport of 50 ,mol/L MeAIB by vesicles prepared from control rats and those prepared from rats treated chronically with GH 12 hours after the last exposure is shown in Figure 5. Uptake was rapid and linear for the first minute under both experimental conditions. Vesicles from animals treated with GH exhibited a decreased rate of an

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TIME (min) FIG. 5. Time course of MeAIB transport by HPMVs from control and growth hormone-treated rats. Rats received 6 mg/kg GH every 12 hours X 4 doses and hepatic plasma membrane vesicles were prepared 12 hours after the last injection of GH. The Na+-dependent transport of 50 ,umol/ L [3H]-MeAIB was determined across time. A representative time course is shown. Data are mean ± SEM and error bars not apparent lie within the symbol.

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Na+-dependent transport at all initial velocities, being decreased 30% at 30 seconds, and peak Na+-dependent accumulation was attenuated 24% at 5 minutes. Equilibrium values were similar in both experimental conditions, indicating vesicles of similar size. Figure 6 depicts a representative kinetic analysis of the reduction in MeAIB transport activity secondary to chronic GH treatment in the rat. At all extravesicular MeAIB concentrations, transport was attenuated in vesicles from GH-treated animals (Fig. 6A). Eadie-Hofstee linear transformation of the data demonstrated that the increase in transport was secondary to a 59% decrease in

Ann. Surg. * September 1992

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FIG. 7. Amino acid transport activity in HPMVs from control and growth hormone-treated rats. Rats received a single dose of growth hormone (6 mg/kg) and hepatic plasma membrane vesicles were prepared 4 hours later. The Na+-dependent transport of 50 Amol/L [3H]-MeAIB or 100 Amol/L [3H]-glutamine by vesicles was determined. Data are mean ± SEM. * p < 0.05. o

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the maximal velocity of transport, with no alteration in transporter affinity (Fig. 6B). In contrast to the effects we observed with chronic GH treatment in both the human and the rat, to investigate the acute effects of GH, a second group of rats was treated with a single injection of GH (6 mg/kg) and vesicles were prepared 4 hours later. Mean transport velocities of 50 Amol/L MeAIB and 100 ,umol/L glutamine demonstrated a trend toward an increase in Na+-dependent MeAIB transport, which did not achieve statistical significance, and a significant 40% increase in Na+-dependent glutamine transport (Fig. 7; p < 0.05). The third GH treatment protocol employed in the rat studies combined the chronic GH treatment of the first group with the 4-hour postinjection time point of the acutely treated second group. Mean Na+-dependent amino acid transport velocities for this third group of rats are shown in Figure 8. As is apparent, no statistically significant differences were found for MeAIB or glutamine transport in this GH-treated group relative to the corresponding control animals, although the activity of MeAIB transport tended to appear diminished.

V/1S] FIG. 6. Kinetic analysis of MeAIB transport activity by HPMVs from control and growth hormone-treated rats. Rats received 6 mg/kg GH every 12 hours X 4 doses and hepatic plasma membrane vesicles were prepared 12 hours after the last injection of GH. (A) The Na+-dependent 10-second transport of [3H]-MeAIB was determined as a function of

increasing extravesicular MeAIB concentration. (B) Regression analysis of the resultant Eadie-Hofstee linear transformation plots revealed that the decrease in MeAIB transport secondary to GH treatment was due to a decrease in maximal transport velocity (Vmax = y-intercept; 235 pmol/mg pro/10 sec in control vs. 97 in GH) with no alteration in transporter affinity (Km = negative slope; 1.5 mmol/L in control vs 1.4 in GH). Representative plots are shown.

Discussion Because surgical patients develop erosion of lean body mass and negative nitrogen balance, efforts have have focused on methods of modifying the stress response in the hopes of improving clinical outcome. Despite attempts to demonstrate otherwise, several reports indicate that aggressive nutritional support does not prevent considerable loss of lean body mass during critical illness. Consequently, attempts have been made to attenuate the cat-

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GROWTH HORMONE AND HEPATIC AMINO ACID TRANSPORT

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FIG. 8. Amino acid transport activity in HPMVs from control and growth hormone-treated rats. Rats received 6 mg/kg GH every 12 hours X 4 doses and hepatic plasma membrane vesicles were prepared 4 hours after the last injection of GH. The Na'-dependent transport of 50 umol/L [3H]-MeAIB or 100 /Amol/L [3H]-glutamine by vesicles was determined. Data are mean ± SEM.

abolic response, employing a combination of hormonal supplementation with nutritional intervention.3'5-7 Many of these investigations have focused on the anabolic agent growth hormone, in large part because of advances in molecular biology, which have made available the genetic vector and technology for the production of relatively large and inexpensive quantities of the compound. Before these advances, therapeutically effective GH was available only from human pituitary extracts and, hence, was reserved for intervention in highly specialized cases, such as pituitary dwarfism. The classical anabolic properties ofGH have been well established. They include nitrogen retention associated with an increase in protein synthesis, and fat mobilization. The nitrogen retention is reflected by a diminished urea excretion that results from a true reduction in hepatic urea production.3'6'7 More recent studies examining the influence of hrGH administration in catabolic patients have demonstrated marked improvements in muscular strength and wound healing.3'5'6"9'20 Much attention regarding the mechanisms underlying this improvement in nitrogen retention has focused on the effects of growth hormone in stimulating protein synthesis and muscle amino acid uptake. Considerably less is known about the role of the liver in nitrogen metabolism during GH therapy. In the current study, we examined the effects of GH on amino acid transport in the liver of healthy patients and in normal rats. We chose to study systems A and N in the liver because of their mediation of the transport of the neutral amino acids alanine and glutamine, respectively, and the relevance ofthese two amino acids in overall nitrogen balance. The role of membrane transport in

359

the regulation of amino acid metabolism has become increasingly apparent in recent years.", 2 22 This is particularly applicable to the liver as the major organ of amino acid metabolism. With reference to ureagenesis, it has been suggested that the presence of hepatic ureagenic enzymes in such great excess indicates that the intracellular availability of amino acid precursor (i.e., concentrative transport) is the rate-limiting factor and strongly influences the flux of nitrogen diverted to ureagenesis.23 In light of the gross observations on nitrogen retention elicited by GH therapy and the accumulating evidence supporting the role of membrane transport as a regulator of hepatic amino acid metabolism, we focused our attention on the hepatocyte plasma membrane in the current investigation. Employing plasma membrane vesicles to assess hepatic amino acid transport activity offers several advantages over other approaches. First, vesicles allow one to examine the inherent amino acid transport activity of the plasma membrane independent of other confounding influences (e.g. intracellular metabolism, substrate delivery, transstimulation/inhibition). Plasma membrane vesicles have been widely employed successfully to obviate these problems by our laboratory and by others in both animal and human studies.'8124-27 Second, vesicles allow the examination of the intrinsic transport activity ofthe hepatocyte in the surgical patient with minimal additional manipulation. Thirdly, it has been demonstrated that membrane transport activity is preserved as it existed in the intact cell during the preparation of vesicles,28 thus allowing an essentially in vivo observation using an in vitro preparation. Because the genes responsible for translation of the System N and System A proteins have not been cloned to date, the characterization and regulation ofthese amino acid transport systems has relied on inhibition studies and physicochemical properties. 8-10222 Amino acid transport system A is a ubiquitous system with a broad neutral amino acid selectivity favoring the small amino acids such as alanine. The N-methylated derivative of aminoisobutyric acid we employed to examine activity (i.e., MeAIB) is the model substrate. System N exhibits a narrow selectivity, favoring the transport of the amino acid amides, such as glutamine, and thus far, has been identified exclusively in hepatocytes.'7 Although the basal activity of system A is considerably lower than that of system N, its ability to transport a much larger number of amino acids than the highly selective N agency should result in a significant decrease in overall amino acid transport into the liver. In general, the effect of GH on amino acid transport is stimulatory in a variety of tissues, including skeletal muscle, heart, diaphragm, adipose tissue, and liver.8"0 In liver, however, these effects have been examined only acutely,

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that is, after a single dose of GH. As early as 1957, Noall et al.29 demonstrated that acute treatment of femae rats with GH resulted in elevated distribution ratios of the nonmetabolizable amino acid analogue aminoisobutyric acid (AIB) in liver within 2 hours. Similarly, Jefferson et al.23 demonstrated that growth hormone administration in vivo to normal or hypophysectomized rats or in the perfusate of an isolated perfused liver preparation increased AIB distribution ratios through a twofold increase in the Vmax of transport within 1 hour. Our vesicle transport data from rats treated acutely with GH for 4 hours before being killed tend to validate at the plasma membrane level these earlier studies indicating a stimulatory action of GH on hepatic amino acid accumulation at the organ level. Interestingly, in both the human and the rat treated chronically by repeated exposure to GH, a very different pattern from the acute response emerged. Chronic GH treatment resulted in a suppression of system A transport activity secondary to a 60% reduction in the transport Vmax whereas system N was unchanged. Thus, chronic treatment reversed the response of system A to acute GH exposure and resulted in a disparate response of system A and system N. Although it is well established that Na+dependent glutamine transport in rat liver occurs almost exclusively by transport system N,'7 available data for human liver have suggested that a significant portion of the mediated uptake of glutamine may occur through system A.'8 We did not observe any inhibition of glutamine uptake by human hepatic vesicles in the presence of MeAIB, and conclude that at physiologic glutamine concentrations the concentrative transfer of glutamine occurs exclusively through system N. The discrepancy between our data and those of Mailliard and Kilberg'8 may be due to the different assay parameters used to evaluate inhibition by MeAIB. Although we employed 100 ,tmol/L glutamine, these authors used 10 ,tmol/L. It is possible that at the much lower glutamine concentration the small amount of glutamine transported by System A represented a much larger fraction of the total glutamine transported and thereby was inhibited to a significant percent in the presence of MeAIB. In addition, the liver we obtained was obtained from patients with normal liver, whereas that obtained by Mailliard and Kilberg'8 was from resected surgical specimens, some ofwhich were removed because of cancer. Moreover, such "normal" liver tissue may have had a period ofhypoxia that occurred during the resection. Our observations on the chronic effect of GH on the activities of systems A and N appear to be a true reflection of the chronicity of GH treatment, and not of the relationship to the time from the last exposure to the hormone. This is evidenced by the similarity of the response in vesicles prepared at the 4-hour timepoint after chronic GH with those prepared at the 12-hour timepoint after

Ann. Surg. * September 1992

chronic treatment. In contrast, treatment with a single dose of GH resulted in a clearcut increase in amino acid transport. Thus, the effect of GH on hepatic amino acid transport appears to exhibit a biphasic response. This is consistent with the observation that GH elicits both early and latent effects in regulating numerous metabolic parameters in a variety of tissues, including liver.23 Further, instances have been noted in which GH-induced stimulation of amino acid transport is transitory and is attenuated after prolonged exposure to the hormone.30 The mechanisms by which multiple GH treatments cause a reduction in system A activity are unclear, but may be related to the effects of GH on insulin handling and insulin-like growth factor-l production.3' The quantity of insulin-like growth factor- receptors in liver is low to undetectable, compared with other tissues. Moreover, preliminary studies with isolated hepatocytes show no effects of GH alone on amino acid transport (Souba, unpublished observations). Thus, the acute stimulation of amino acid transport by GH in vivo or in HPMVs may involve insulin or possibly glucocorticoids. Consistent with this possibility is the observation that chronic GH treatment causes insulin resistance, apparently secondary to a GH-induced insulinemia and the consequent downregulation ofinsulin receptors. In hepatocytes, such a GHinduced insulin receptor deficit could reduce system A activity, which has been shown to be regulated by insulin (and by glucocorticoids).8-'0 Whether chronic GH treatment inhibits hepatic amino acid transport System A directly or indirectly, one would expect this alteration at the membrane level to be reflected at the organ level. Welbourne et al.7 examined hepatic amino acid fluxes in hypophysectomized and hypophysectomized chronically GH-supplemented rats. Although not statistically significant, they observed a 30% decrease in hepatic alanine extraction (p = 0.08) and a concomitant increase in glutamine uptake at the whole organ level, alterations that occurred in the face of no change in the circulating concentrations of these amino acids and no change in hepatic bloodflow. These observations are consistent with regulation at the level of plasma membrane transport and the data we have reported here. Welbourne and colleagues7 also observed that a decrease in ureagenesis was associated with a shunting of glutamine nitrogen away from its usual incorporation into urea and toward hepatic glutamate release. Taken together with our data demonstrating divergent regulation of membrane transport, these studies suggest that the hepatocyte shunts nitrogen away from ureagenesis in response to chronic GH by either a reduction in the intracellular availability of amino acid nitrogen (by decreasing the activity of plasma membrane transport, as appears to be the case for the System A substrate alanine), or by modifying intracellular metabolism (by shunting nitrogen into glutamate and

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GROWTH HORMONE AND HEPATIC AMINO ACID TRANSPORT

from urea production as appears to be the case for glutamine). Through either mechanism, these studies demonstrate that the liver is a not simply a passive participant in the overall improvement in nitrogen balance that accompanies GH administration, but is instead an active organ that may redistribute amino acid nitrogen to peripheral tissues. Studies designed to evaluate the effects of GH treatment on hepatic amino acid transport during catabolic states when amino acid uptake is markedly increased are in progress. away

References 1. Cuthbertson DP. Physical injury and its effects on protein metabolism. In Munro HN, Allison JB, eds. Mammalian Protein Metabolism. New York: Academic Press, 1964; 373-414. 2. Wilmore DW. The Metabolic Management ofthe Critically Ill. New York: Plenum, 1977. 3. Jiang ZM, He GZ, Zhang SY, et al. Low-dose growth hormone and hypocaloric nutrition attentuate the protein-catabolic response after major operation. Ann Surg 1989; 210:513-525. 4. Streat SJ, Beddoe AH, Hill GL. Aggressive nutritional support does not prevent protein loss despite fat gain in septic intensive care patients. J Trauma 1987; 27:262-266. 5. Wilmore DW. Catabolic illness. Strategies for enhancing recovery. N Engl J Med 1992; 325:695-702. 6. Ziegler TR, Young LS, Manson JM, Wilmore DW. Metabolic effects of recombinant human growth hormone in patients receiving parenteral nutrition. Ann Surg 1988; 208:6-16. 7. Welbourne T, Joshi S, McVie R. Growth hormone effects on hepatic glutamate handling in vivo. Am J Physiol 1989; 257:E959-E962. 8. Christensen HN, Kilberg MS. Amino acid transport across the plasma membrane: role of regulation in interorgan flows. In Yudilevich DL, Boyd CAR, eds. Amino Acid Transport in Animal Cells. Manchester, UK: Manchester University Press, 1987; 10-45. 9. Kilberg MS. Amino acid transport in isolated rat hepatocytes. J Membr Biol 1982; 69:1-12. 10. Shotwell MA, Kilberg MS, Oxender DL. The regulation of neutral amino acid transport in mammalian cells. Biochim Biophys Acta 1983; 737:267-284. 11. Prpic V, Green K, Blackmore P, Exton J. Vasopressin, angiotensin II, alpha-adrenergic-induced inhibition of Ca++ transport by rat liver plasma membrane vesicles. J Biol Chem 1984; 259:13821385. 12. Moore DJ. Enzyme purification and related techniques. Meth Enzymol 1971; 22:130-148. 13. Swanson MA. Glucose-6-phosphatase from liver. Methods Enzymol 1955; 2:541-543. DISCUSSION DR. DOUGLAS W. WILMORE (Boston, Massachusetts): Thank you, Mr. President. The plasma amino acid pool is drawn on by visceral organs and is fed primarily by the skeletal muscle. Thus, in normal adults the plasma amino acid pool represents the balance between amino acids released from the periphery and what is taken up by visceral organs, primarily the liver. This is not so in growing children, for amino acids are taken up in a net sense by skeletal muscle as the child grows. And as we start talking more and more about growth hormone administration in adults, we really must think about the growing adult, because now plasma amino acids are being taken up by skeletal muscle under these conditions. If both liver and skeletal muscle are competing for amino acids, then the levels of amino acids in the bloodstream may decrease, to very low levels. What is presented in this paper is a mechanism that can regulate this process so that skeletal muscle can take up amino acid and can grow and at the same the liver can dampen its uptake of amino acid uptake

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14. Kilberg MS, Christensen HN. Electron transferring enzymes in the plasma membrane of the Ehrlich ascites tumor cell. Biochem J 1979; 18:1525-1530. 15. Fiske CH, Subbarow Y. The colorimetric determination of phosphorus. J Biol Chem 1925; 66:375-400. 16. Bensadoun A, Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem 1976; 70:241-250. 17. Kilberg MS, Handlogten ME, Christensen HN. Characteristics of an amino acid transport system in rat liver for glutamine, asparagine, histidine, and closely related analogs. J Biol Chem 1980; 255:4011-4019. 18. Mailliard ME, Kilberg MS. Sodium-dependent neutral amino acid transport by human liver plasma membrane vesicles. J Biol Chem 1990; 265:14321-14326. 19. Wilmore DW, Moylan JA, Bristow BF, et al. Anabolic effects of human growth hormone and high caloric feedings following thermal injury. Surg Gynecol Obstet 1974; 138:875-883. 20. Herndon DN, Barrow RE, Kunkel KR, et al. Effects of recombinant growth hormone on donor-site healing in severely burned children. Ann Surg 1990; 212:424-431. 21. Christensen HN. Role of amino acid transport and countertransport in nutrition and metabolism. Physiol Rev 1990; 70:43-77. 22. Collarini EJ, Oxender DL. Mechanisms of transport of amino acids across membranes. Annu Rev Nutr 1987; 7:75-90. 23. Jefferson LS, Schworer CM, Tolman EL. Growth hormone stimulation of amino acid transport and utilization by the perfused rat liver. J Biol Chem 1975; 250:197-204. 24. Pacitti AJ, Austgen TR, Souba WW. Adaptive regulation of alanine transport in hepatic plasma membrane vesicles (HPMVs) from the endotoxin-treated rat. J Surg Res 1991; 51:46-53. 25. Pacitti AJ, Copeland EM, Souba WW. Stimulation of hepatocyte System y+-mediated L-arginine transport by an inflammatory agent. Surgery 1992 (in press). 26. Jacob R, Rosenthal N, Barrett EJ. Characterization of glutamine transport by liver plasma membrane vesicles. Am J Physiol 1986; 251 :E509-E514. 27. Rosenthal NR, Jacob R, Barrett E. Diabetes enhances activity of alanine transport in liver plasma membrane vesicles. Am J Physiol 1985; 248:E581-E587. 28. Schenerman MA, Kilberg MS. Maintenance ofglucagon-stimulated System A amino acid transport activity in rat liver plasma membrane vesicles. Biochim Biophys Acta 1986; 856:428-436. 29. Noall MW, Riggs TR, Walker LM, Christensen HN. Endocrine control of amino acid transfer. Science 1957; 126:1002-1005. 30. Ahren K, Albertson-Wikland K, Isaksson 0, Kostyo JL. Cellular mechanisms of the acute stimulatory effect of growth hormone. In Pecile A, Muller EE, eds. Growth Hormone and Related Peptides. Amsterdam: Excerpta Medica, 1976. 31. Daughaday WH, Rotwein P. Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum and tissue concentrations. Endocr Rev 1989; 10:68-91. and maintain the plasma amino acid pool. Thus, the concentrations of plasma amino acids in these individuals would be important, and I would ask the authors if they have these data. I also would like to make some comments about the dose of growth hormone used. Almost all the doses that are being used now are in the pharmacologic range. This is not physiologic replacement. The average dose we use is about 0.13 mg/kg; that is not quite in the middle of the 0.1 to 0.2 mg/kg doses used in this study. Real effects were only seen with the 0.2-mg/kg dose, and this raises questions about whether the 0.1-mg/kg dose given for longer periods would have similar effects. Would the authors also comment about standardization of food intake in the individuals? Was anesthesia standardized in these patients and could type of anesthesia change amino acid transport? Finally, if you took liver slices and added growth hormone in vitro, could you induce similar changes in transport? If not, can you give insulin-like growth factor I to in vitro slices and also induce these changes? This is important information because it expands our knowledge about the mechanisms of hormonal manipulations we will be using in the future care of our patients.