Integrated nutritional, hormonal, and metabolic effects of recombinant human growth hormone (rhGH) supplementation in trauma patients

APPLIED NUTRITIONAL INVESTIGATION

Nutrition Vol. 12, Nos.

11112, 1996

Integrated Nutritional, Hormonal, and Metabolic Effects of Recombinant Human Growth Hormone (rhGH) Supplementation in Trauma Patients MALAYAPPA

JEEVANANDAM, PHD, NANCY J. HOLADAY, AND SCOTT R. PETERSEN, MD, FACS

BS,

From the Trauma Center, St. Joseph’s Hospital and Medical Center, Phoenix, Arizona, USA Date accepted: 11 April 1996

ABSTRACT

An anabolic stimulus is needed in addition to conventional nutritional supportin the catabolic “flow” phase of severe trauma. One promising therapy appears to be rhGH infusion which has direct as well as hormonal mediated substrate effects. We investigated on a whole-body level, the basic metabolic effects of trauma within 48-60 h after injury in 20 severely injured (injury severity score [ISS] = 31 ? 2), highly catabolic (N loss = 19 + 2 g/d), hypermetabolic (resting energy expenditure [REE] = 141 + 5% basal energy expenditure [BEE]), adult (age 46 f 5 y) multiple-trauma victims, before starting nutrition therapy and its modification after 1 wk of rhGH supplementation with TPN ( 1.1 x REE calories, 250 mg N *kg-’ *d-l). Group H (n = 10) randomly received at 8:00 a.m. on a daily basis rhGH (0.15 mg *kg-’ *d-’ ) and Group C (n = 10) received the vehicle of infusion. Protein metabolism (turnover, synthesis and breakdown rates, and N balance) ; glucose kinetics (production, oxidation, and recycling) ; lipid metabolism, (lipolysis and fat oxidation rates), daily metabolic and fuel substrate oxidation rate (indirect calorimetry); and plasma levels of hormones, substrates, and amino acids were quantified. In group H compared to group C: N balance is less negative ( -41 t 18 vs - 121 5 19 mg N. kg-’ -d-l, P = 0.001); whole body protein synthesis rate is 28 ? 2% (P = 0.05) higher; protein synthesis efficiency is higher (62 + 2% vs 48 + 3%, P = 0.010); plasma glucose level is significantly elevated (256 5 25 vs 202 + 17 mg/ dL, P = 0.05 ) without affecting hepatic glucose output ( 1.5 1 2 0.20 vs 1.56 t 0.6 mg N *kg-’ *min-’ ), glucose oxidation and recycling rates; significantly enhanced rate of lipolysis (P = 0.006) and free fatty acid reesterification (P = 0.05); significantly elevated plasma levels of anabolic GH, IGF-1, IGFBP-3, and insulin; trauma induced counter-regulatory hormone (cortisol, glucagon, catecholamines) levels are not altered; trauma induced hypoaminoacidemia is normalized (P < 0.05) and 3-methylhistidine excretion is significantly low (P < 0.001). Improved plasma IGF-1 levels in Group H compared with Group C account for protein anabolic effects of adjuvant rhGH and may be helpful in promoting tissue repair and early recovery. Skeletal muscle protein is spared by rhGH resulting in the stimulation of visceral protein breakdown. The hyperglycemic, hyperinsulinemia observed during rhGH supplementation may be due to defective nonoxidative glucose disposal, as well as inhibition of glucose transport activity into tissue cells. The simultaneous operation of increased lipolytic and reesterification processes may allow the adipocyte to respond rapidly to changes in peripheral metabolic fuel requirements during injury. This integral approach helps us to better understand the mechanism of the metabolic effects of rhGH. OElsevier Science Inc. 1996 Nutrition 1996; 12:777-787

,

Key words: acute trauma injury effects, growth hormone treatment, nutrition and trauma

Presented at the 17th Congress of ESPEN held in Rome, Italy, September lo- 13, 1995. Supported in part by a grant from NIH (GM46548) and a grant (82-2688) from the Arizona Disease Control Research Commission. Correspondence to: M. Jeevanandam. PhD, Trauma Center, St. Joseph’s Hospital and Medical Center. 3.50 West Thomas Road Phoenix, AZ 85013, USA.

Nutrition 12:777-787, 1996 OElsevier Science Inc. 1996 Printed in the USA. All rights reserved.

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0899-9007/96/$15.00 PII: SO899-9007(96)00220-l

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INTRODUCTION

Trauma is the leading cause of death during the first four decades of life, in the U.S., and slow in recovery with many pitfalls along the course of clinical management. At the onset of trauma, the metabolic machinery is geared up to meet the injury-induced challenges associated with a crisis of energy availability. The early catabolic “flow” phase of severe injury is characterized by many changes including hypermetabolism with increased resting energy expenditure (REE), enhanced glucose and lipid kinetics, hypercatabolism with increased mobilization and loss injury of nitrogen, hyperglycemia with enhanced glucose production and glucose intolerance, increased secretion of counter-regulatory hormones, and normal or hyperinsulinemia with decreased effectiveness of stimulated insulin production. Hypoaminoacidemia, in both intracellular and extracellular compartments, is also a common finding of severe injury. Similar to most critical illnesses, initial observations and treatment of severely traumatized patients influence their ultimate outcome. The combination of decreased nutritional intake and increased nutrient demand by tissues and organs in the early “flow” phase of acute injury emphasizes the importance of nutritional management of traumatized patients. The ultimate goal for nutritional support in trauma is to increase protein anabolism, mihimize protein catabolism, and mobilize fat fuel sources. This may result in better utilization of the substrates of protein, fat, and carbohydrate metabolism. Current intravenous nutritional support techniques may prevent further breakdown of protein but will not by themselves stimulate protein synthesis significantly or produce noticeable accrual of protein during short-term use.r,’ The addition of a potent anabolic stimulus during intensive nutritional therapy appears to be a reasonable adjuvant for the minimization of muscle mass erosion.’ Development of an acute deficiency in the circulatory levels of anabolic growth hormone (GH) and insulin-like growth factor-l (IGF-1) in multiple-trauma victims during the early catabolic flow phase of severe injury has been reported.3.4 The promising adjuvant therapy seems to be the GH infusion’-’ which has direct as well as indirect hormonal mediation and substrate effects. An integrated attempt to derive the mechanisms for the nutritional, hormonal, and metabolic effects of recombinant human growth hormone (rhGH) is reported in this study. Parts of this investigation had been published separately elsewhere 6-9 and here they are consolidated with additional data (daily excretion of 3-methylhistidine [3-MHS], erotic acid, uric acid, and hydroxyproline [OHPR]) to better understand the mechanisms of the metabolic efficacy of rhGH in the utilization of body energy stores which may lead to early recovery from injury. MATERIALS

AND METHODS

Subjects

Twenty severely injured adult patients (17 men and 3 women) were studied after admission to the Intensive Care Unit of the Level I Trauma Center at St. Joseph’s Hospital and Medical Center in Phoenix, Arizona. The protocol was reviewed and approved by the Institutional Review Board. Written informed consent was obtained following explanation of the study to the patient or legal representative. Relevant patient characteristics on admission are given in Table I and the indi-

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vidual diagnoses were reported previously.6 The studies were initiated within 48-60 h after the patients sustained multiple injuries, when they were receiving maintenance fluids and electrolytes but no calories or nitrogen. The patients were evaluated and resuscitated according to their individual needs. None of the patients were septic or had diabetes mellitus, liver dysfunction, renal insufficiency, malignant disease, or multiple organ failure. They appeared to be well nourished prior to injury and none were chronically ill. On admission, the injury severity was determined by the Injury Severity Score (ISS) based upon the Abbreviated Injury Score of the three most serious anatomic injuries.” All had at least one major injury and multiple minor injuries with ISS ranging from 17 to 50 with a mean of 31 + 3. All patients required ventilator support during the basal study (fraction of inspired oxygen ~0.40) and 4 patients were weaned off mechanical ventilation during the 7 d of nutritional therapy. Most ( 14 of 20) of the patients were involved in motor vehicle crashes and sustained multiple skeletal fractures with soft-tissue injuries. Four patients were victims of penetrating wounds to the abdomen, chest, or face; one patient had an accidental fall resulting in multiple fractures; and another patient was admitted with a closed head injury and multiple fractures after his experimental airplane crashed into a tree. Experimental

Study Protocol

Twenty-four hour urine collections through a Foley catheter were initiated and continued until the end of the study. When the medical status of the patients became stable and resuscitation was complete, a blood sample was drawn from each patient in the morning through an existing arterial line for basal substrate and hormone measurements. This occurred 48-60 h after injury during the early stages of the catabolic phase of severe injury. The patients were weighed (Flexicair MC3, Support System, Inc., Charleston, SC, USA) in the morning and the daily weights were recorded. They were randomized to receive (Group H) or not to receive (Group C) rhGH during nutritional support by total parenteral nutrition (TPN). Gas Exchange

Indirect

Calorimetry

Oxygen consumption (VO,) , carbon dioxide production (Vco,), and respiratory quotient (RQ) were measured using the metabolic cart (Horizon Metabolic Measurement Cart, Sensormedics, Anaheim, CA, USA). All the patients were on ventilator support and the exhaled flow was directly connected to the metabolic cart. The instrument was calibrated before each measurement and the stability of the instrument conditions was observed for at least a lo-min equilibration period. Following the stabilization period, the test measurement was performed over 29 min of continuous sampling. One-minute averages of VOZ, VCO~, and RQ were calculated. Means of VO, and VcoZ during the 20-min period along with the urinary total nitrogen excretion were used to calculate REE and substrate oxidation rates.“.” Predicted basal energy expenditure (BEE) was calculated by the appropriate Harris-Benedict equation, taking into consideration age, gender, height, and weight.13 Glycerol Turnover Study

At 7:00 a.m., a two-stage primed constant infusion of glycerol (10% wt/wt) was started to measure the glycerol turnover rate.14 The net rate of glycerol turnover in plasma reflects the net unidirectional whole-body lipolysis rate (WBLR) .14,15The

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details of this lipid study were reported previously.’ At the end of each stage, three blood samples (2 mL each) were drawn at equal intervals for glycerol measurements. Glucose Kinetic Study At 10:00 a.m.. a primed-constant infusion of sterile and nonpyrogenic (6-7H) glucose and ( U-14C) glucose (ICN Radiochemicals, Irvine, CA, USA) in normal saline was administered to all subjects for 130 min. After a bolus priming dose over a 2-min period, the continuous infusion followed. The infusion rates of each of the isotopic glucose solutions during this period were 2.0 nCi * kg-’ - min-‘. The priming dose to infusion rate ratio was 80:1 for each isotope.16 The bicarbonate pool was primed with 60 nCi/kg of NaH’4C03.‘7 During this constant continuous infusion of isotopes, arterial blood samples (5 mL each) were drawn at 110, 120, and 130 min to determine the specific radioactivities of the isotopic glucoses. The expired gas was collected at 5-min intervals ( 105- 110, 115- 120, and 125-130) in plastic bags for specific activity of CO2 in the breath gas. The experimental procedures did not interfere with patient care. None of the subjects exhibited glucosuria during this study. The details of the glucose study were reported previously.’ Whole-Body

Protein Kinetic St&y

After the end of the glucose study at about 12:30 p.m., the protein turnover measurements were started using a primed constant infusion of 15Nglycine and periodic measurements of 15Nexcretion in urinary urea and ammonia.‘8 Urine specimens were collected at 12, 16, 18, 20, 22, and 24 h for the measurements bf “N in urea and ammonia. The details of this study were reported previously.’ At the end of this study period where basal fat, glucose, and protein kinetics were measured (Study I) in each patient, nutritional support was started and continued for 7 d with or without rhGH bolus administration. Nutritional

Support

At the end of Study I, intravenous feeding (TPN) with the necessary electrolytes, minerals, trace elements, and vitamins administered via a central venous catheter was initiated and continued for 7 d. All patients were given continuous infusion of nutrients at a constant rate for the duration of the study. The TPN diet contained 250 mg N * kg-’ * d-’ as commercially available balanced free amino acid (AA) mixture ( 10% Aminosyn, Abbott Laboratories, North Chicago, IL, USA) and the energy requirements were based on 1.1 X REE. Nonprotein calories were provided as dextrose. To prevent essential fatty acid deficiency, 500 mL of 10% lipid emulsion (Intralipid lo%, Kabi Vitrum, Alameda, CA, USA) was given intravenously over an 8-h period on Day 3. During this nutritional therapy, the patients were randomized to receive (Group H. II = 10) or not io receive (Group C, II = 10) intramuscular rhGH (Somatropin, Genentech, Inc., San Francisco, CA, USA) every day at 8 a.m. (after morning blood sampling) at a dose of 0.15 mg *kg-’ -d-l. This dose of rhGH was chosen because it had been shown to induce adequate metabolic and hormonal activities. 5,‘9.‘oTwenty-four hour urine collection for daily N balance and renal function, and morning blood samples for BUN and hormonal measurements were continued. All patients tolerated the intravenous nutritional regimen and had an uncomplicated course during this period. Repeat Kinetic Studies (Study II)

At the end of 7 d of continuous feeding, the fat, glucose and protein kinetic studies and gas exchange measurements

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779

were repeated as described previously while the TPN was continued. Study II was intended to document the effects of rhGH in modifying the catabolic state of injury during the flow phase with adequate nutritional support. Chemical Methods and Calculations

Using validated methods and established procedures, the protein,6 fat,7 and glucose’ kinetic parameters and plasma free amino acid levels’ were determined as reported by us previously. 3-Methylhistidine (3-MHS) is a noninvasive marker of myofibrillar protein breakdown and its urinary excretion is a valid index of skeletal muscle breakdown in humans not eating meat for at least 48-60 h. Excreted 3-MHS in the daily urine samples collected in this study was determined by the automated ion exchange method using an amino acid analyzer (Model 7300; Beckmann Instruments, Palo Alto, CA, USA) after deproteinization with sulfosalicylic acid. Single buffer, short program was used with (Y amino /? guanidinopropionic acid as an internal standard. When collagen is broken down, OHPR is released and largely excreted unchanged. Urinary total OHPR was measured, after acid hydrolysis, in an amino acid analyzer using a single buffer, short column and glucosaminic acid as the internal standard. Orotic acid is a normal intermediate of pyrimidine biosynthesis. Elevated urinary erotic acid had been proposed to result from decreased urea cycle capacity and impaired urea synthesis. Orotic acid excretion could be used as a method of determining in vivo amino acid imbalance in patients receiving TPN. The calorimetric method of Harris and Oberholzer” as modified22,2’ by us was used to measure erotic acid in urine samples. This assay procedure was previously validated by using HPLC2’ Uric acid is measured by standard procedure with a Micro Centrifugal Analyzer (Multistat Plus, Instrumentation Laboratory, Lexington, MA, USA). Statistics

Values in the text are given as the mean 2 SEM. Since two studies were performed in each patient, Student’s paired t test was used to estimate the differences between variables. Analysis of variance for repeated measures was used to test differences between treatment and control groups. Correlation coefficients were calculated by linear regression.” A P value of ~0.05 was considered statistically significant. RESULTS Nutritional effects on the energy fuel kinetics of severely injured patients were studied, once in the basal condition before the initiation of nutritional therapy and again after 7 d of adequate nutritional support with or without daily rhGH supplement. Clinical characteristics of the enrolled trauma patients are given in Table I and the individual diagnoses were reported previously.6 The altered plasma substrates, hormonal parameters, and free amino acid levels due to intravenous nutrition with (Group H) or without (Group C) adjuvant rhGH are given in Tables II, III, and IV, respectively. Daily urinary excretion data is summarized in Table V. Group H and Group C patients have similar body weight (80 2 6 kg vs 83 ? 4 kg), body mass index (27 +- 2 vs 28 ? 2 kg/m’) and ISS (30 ? 4 vs 31 ? 2), and are equally hypermetabolic (REE = 38 2 8 and 44 ? 6% higher than their predicted BEE), and highly catabolic (daily N excretion = 18 ? 3 and 20 f 3 g, respectively). The types of injuries, blunt or penetrating, were equally distributed between the two groups.

780

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Plasma Substrate and Hormone

Levels

Trauma-induced hyperglycemia is further exaggerated with TPN but significantly more with rhGH supplementation. Both groups of trauma patients are hypoalbuminemic and nutrition with or without rhGH cannot improve the albumin or total plasma protein status. There are no significant changes due to rhGH supplementation in many of the plasma substrates like lactate, glycerol, TA, free fatty acids (FFA) and ketone bodies. The plasma levels of GH and IGF- 1 in the basal trauma conditions are significantly (P = 0.05) low, compared with the reported3,4 normal values in healthy subjects. Although pulsatile GH secretion persists in injured patients, mean 8 a.m. GH concentration is not different from 24-h integrated GH concentrationz5 Provision of nutrients alone without rhGH increases the IGF-I levels by 93% from the basal; however, with rhGH they are raised by 210%. This indicates the stimulation of IGF-1 secretion by exogenous GH. Plasma GH level is doubled in the control group with TPN alone for 7 d, but with additional exogenous rhGH it is raised by 730%. Increased plasma levels of GH would potentially change the level of IGF- 1 in the circulation, the delivery of IGF-1 to the target tissues, and its biological effectiveness. Adjuvant rhGH significantly improves the levels of the major binding protein of IGF-1 (IGFBP-3). This improvement indicates more bioavailability of free IGF-1 for anabolic responses. Nutrition with or without adjuvant rhGH has no effect on the counter-regulatory hormones, cortisol, glucagon, and catecholamines. However, insulin levels are significantly more increased in rhGH patients. This hyperinsulinemia occurs with hyperglycemia due to rhGH. Plasma Amino Acid Levels

Hypoaminoacidemia of acute trauma is confirmed in both groups of patients. Seven days of TPN alone could not restore the normal levels of total amino acids (TAA) but with additional provisions of rhGH, the AA levels are normalized. The decrease in TAA levels in trauma patients is mainly due to decreases in many of the nonessential amino acid levels. The branched-chain amino acid (BCAA) levels are kept remarkably constant with nutritional therapy, although infusion of rhGH tended to increase BCAA, especially valine. The plasma phenylalanine concentration is increased in both groups of traumatized patients, compared with normal subjects, in the basal state and also during nutritional therapy with or without rhGH. Alanine and glycine levels are decreased by 50% because of trauma and they are restored only in patients receiving rhGH. Similarly, serine levels are decreased by 35% because of trauma and could be restored only by rhGH treatment. The characteristic decrease in plasma glutamine levels in traumatized patients could not be restored even with rhGH supplementation, although that group showed a better improvement. The decreased arginine and omithine levels caused by the trauma are restored in both groups of patients; however, they are significantly increased in rhGH patients. Parameters

of Protein Metabolism

Provision of intravenous nutrients for 7 d with daily rhGH infusion significantly improves the N balance ( -41 + 18 mg N * kg-’ * d-’ for patients receiving rhGH and - 13 1 ? 14 mg N-kg-’ * d-’ for patients without rhGH; P 5 0.01). This improvement in N retention is also reflected in the significantly low BUN (15.2 2 2.3 vs 22.4 ? 1.5 mg/dL; P = 0.025) and low urinary urea excretion (13.5 ? 1.0 vs 21.2 I 1.1 gN/d; P = 0.02) in the rhGH group. Creatinine originates in the muscle and the amount of creatinine excreted in the urine is

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proportional to the body muscle mass.26 There is a tendency (not significant) toward decreased creatinine excretion in traumatized patients due to intravenous feeding. Urinary excretion of 3-MHS could be used as an accurate, albeit indirect, determinant of muscle myofibrillar protein degradation in humans.27-29 Daily 3-MHS excretion in trauma patients is illustrated in Fig. 1. There is a statistically significant (P < 0.001) inverse relationship between daily nitrogen balance (X, mg N -kg-’ * d-‘) and 3-MHS excretion (Y, pmol/d), (Y = 198 - 1.62X, r = -0.65) in these patients (Fig. 2). Patients with rhGH supplementation excreted significantly less 3-MHS (204 ‘_ 28 vs 431 ? 29 pmol/d, P 5 O.OOl), on day 5, 6, and 7, indicating less muscle protein breakdown during TPN. Molar ratio of 3-MHS/creatinine is also significantly low (0.0217 C 0.0022 vs 0.0310 + 0.0015; P 5 0.05) in rhGH group. This also reflects the lower amount of muscle protein breakdown over the amount of muscle mass. Daily mean 3MHS excretions could be converted to muscle protein equivalents using the relationship of 4.2 pmol of 3-MHS/g mixed muscle protein in humans. ‘* This result is compared to whole body protein breakdown rate in Table VI. Contribution of muscle protein breakdown to whole-body protein is significantly reduced from 36 of: 5% to 23 ? 3% in rhGH patients, whereas it remains the same in the patients without rhGH. Since OHPR is a specific indicator of collagen disposition and a good marker of bone breakdown, the beneficial effects of GH on wound healing were studied with the measurement of OHPR excretion. There is a significant rise in urinary excretion of OHPR in both groups of patients due to TPN with no significant difference between the groups. Mild erotic aciduria could be used as a method of determining in vivo amino acid imbalance and urea production.22.23 Basal erotic acid excretion is not changed due to TPN with or without rhGH. Uric acid is the end product of purine metabolism in humans. Hypouricemia and mild uricosuria are the basal-trauma responses.23 TPN with or without rhGH significantly reduces daily uric acid excretion showing no specific response due to rhGH. The results of whole-body protein kinetics are summarized in Table VII. Under basal conditions,, the trauma patients show a significant increase in both protein breakdown (36%) and synthesis rate (26%) compared with uninjured normal subjects. The utilization efficiency of the mobilized body proteins recycled to synthesize new proteins (protein synthesis efficiency [PSE]) in the absence of exogenous N intake (Study I) is similar in both groups of patients (64.2 t 3.6% and 65.3 + 3.0%). These values are significantly lower than seen in fasting normal subjects (76.3 2 3.3%) .30During protein intake (Study II) both the exogenous and endogenous fluxes are considered.30 Provision of amino acids as TPN in normal subjects lowers this total PSE by 21% to 60.1 ? 2.6%; in trauma subjects the PSE is similarly lowered by 26% from 65.3 + 3.3% to 48.0 ? 2.8%. When the trauma subjects were given adjunctive rhGH, however, PSE remained unchanged from baseline values (64.2 IT 3.6 to 62.3 ? 2.2), showing a better utilization of the exogenous supply of amino acids to synthesize new proteins. In normal subjects, intravenous nutrition lowers the protein breakdown rate by 57% compared with 28% and 31% in trauma victims with and without rhGH respectively. Improved N retention in intravenously fed trauma victims with rhGH thus seems not to be due primarily to a reduction in protein breakdown rate but to maintaining a higher protein synthesis rate and PSE. Parameters

of Glucose Metabolism

The effects of nutrition on the kinetic parameters of glucose

production and utilization are given in Table VIII. In the basal

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TABLE I. ADMISSION CHARACTERISTICS OF TRAUMA PATIENTS (STUDY 1) No. and sex Age (Y) Weight (kg) BMI (kg/m’) ISS

RQ REE (% BEE) N loss (gN/d) N loss (mg N* kg-’ . d-‘)

17Ml3F 46.3 + 5.0 83.4 2 6.0 27.2 t- 1.2 30.6 ? 2.1 0.74 2 0.02 1.41 2 5 19.2 + 2.2 214 2 15

production is suppressed significantly and still holds a significant (r = 0.68; P = 0.005) linear relationship with the plasma glucose appearance rate. This amount of glucose intake (4.7 -C 0.5 mg glucose * kg-’ *min-’ ) could not completely suppress the endogenous hepatic glucose production, confirming the previous reports from septic3’ and injured” patients. The extent of glucose recycling through three carbon fragments under the basal conditions (12%) is similar to that reported (9.8%) in trauma subjects.33 During TPN, with or without rhGH supplementation, the extent of glucose recycling remains unchanged. Parameters of Lipid Metabolism

Mean + SEM. BMI. body mass index; ISS, injury severity score; RQ, respiratory quotient; REE, resting energy expenditure: BEE. basal energy expenditure.

study there is no significant difference between the two groups of trauma patients. The slightly elevated glucose production rate at base line (3.5 t 0.2 vs 4.4 ? 0.7; P = NS) is due to

higher values (9.39 and 6.66) in two control patients. Both groups have received equivalent amounts of glucose during the second study. Although TPN increases plasma glucose level, administration of rhGH further enhances (P < 0.05) the hyperglycemia significantly. Glucose clearance in Group C patients is increased from a basal value of 2.9 ? 0.3 to 3.8 ? 0.3 mL * kg-’ - min-’ (P I 0.05) during TPN, an increase of 31%. However, there is no change in Group H patients (2.5 -+ 0.2 to 2.7 ? 0.2). The groups with or without adjuvant growth hormone are similar in suppressing (45%) hepatic glucose output, increasing Vco2 (30%) and increasing glucose oxidation (140%). There is no significant correlation between day 7 (Study II ) GH or IGF- 1 concentrations in plasma and absolute glucose oxidation rates. Hepatic glucose output is similar in both groups of patients in Studies I and II and the percent glucose recycling is also similar in the two groups. Under basal conditions (Study I) plasma appearance rate is the same (by definition) as the rate of hepatic glucose production since there is no exogenous intake. During TPN (Study II) the endogenous

The effects of nutrition on the kinetic parameters of lipid mobilization and utilization are given in Table IX. There is no significant change in the already elevated initial REE, and the patients are still hypermetabolic with enhanced VOp. The change in RQ reflects the preferential use of fat in Study I and of the mixed substrate fuel in Study II. The RQ has not exceeded 1.00 in any of the patients during nutritional support, showing the absence of net fat synthesis. Net fat oxidation accounts for about three-quarters of the REE in Study I, which falls to 40% of REE in TPN-fed patients and further to 25% when supplemented with rhGH. This adaptive low-fat oxidation due to adjuvant rhGH results in a trend toward increased glucose and decreased protein oxidation rates. Glycerol turnover rate, which reflects the WBLR, is decreased by 31% due to glucose-based TPN (Group C) . However, when supplemented by rhGH (Group H), it is significantly increased by 18%. This increase in lipolytic rate due to rhGH supplement is statistically significant (P = 0.006, ANOVA). The elevated plasma glycerol level in Study II of Group H patients seems to be due to increased lipolysis with no change in metabolic clearance. Lipolysis of body triglyceride (TG) stores results in FFA and glycerol, and the mobilized FFA will be either oxidized to CO? or used in the resynthesis of TG. The nutritional effect with or without rhGH in the injured patients shows similar trends in plasma FFA (57-60% down), TG (37-31% up), and ketone body (72-93% down) levels (Table II). Nutritional support increases the reesterification rate of the hydrolyzed TG, which is more significant with rhGH supplementation (68 vs 43% ). The energy cost associated with this TG/FFA cycling is calcu-

TABLE II. CIRCULATING PLASMA SUBSTRATE LEVELS DUE TO ADJUVANT rhGH Trauma: Day 0 Substrates Glucose (mg/dL) BUN (mg/dL) Lactate (mg/dL) Glycerol @mol/L) TG (mg/dL) FFA (pmol/L) Albumin (g/L) Total Protein (g/L) Ketone Bodies (pmol/L)

Group H 138 * 16 2 7.7 2 74 t102 5 625 -c 26~ 43 t 669 +

9t 3 1.0 II 16 135

I 3 209t

Trauma: Days 5, 6. and 7 (average) Group C 150 15 7.6 71 140 450 26 46 245

_f 137 + 2t ? 1.0 2 11 c 19 2 45 _f 2 2 2 + 827

Mean + SEM. FFA, free fatty acids; rhGH, recombinant human growth hormone; TG, triglycerides. * P s 0.05 vs patients Group C (no rhGH) for that day. t P c 0.05 vs days 5, 6, 7 (average).

Group H

Group C

256 2 20* 16 + 1* 8.1 i 1.0 862 16 140 ? 23 269 t 42 22 t 11 47 i_ 2 45 i 1

202 f 17 22 2 2 7.9 * 1.4 59 ? 7 183 2 35 180 -c 40 2.5 +- 2 55 t 3 68 + 18

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TABLE III. CIRCULATING PLASMA HORMONAL PROFILE DUE TO ADJUVANT rhGH IN TRAUMA PATIENTS Trauma: Day 0 Normals* (uninjured)

GH (ng/mL) IGF-I (ng/mL) Insulin (~IU/mL) IGFBP-3 (ng/mL) C-Peptide (ng/mL) c0rtis01 (pg/dL) Glucagon (pg/mL) Epinephrine (pg/mL) Norepinephrine (pg/mL)

2.92 228 7.5 2885 1.8 14

+ ? + ? ? t 90 f. 223 c

Group H +rhGH

0.93 21 0.67 205 0.2 2

1.18 74 12.9 1216 2.7 23 61 223 816

20 59

t2 rt 2 ? 2 ” ? 2

Trauma: Days 5, 6, and 7 (average) Group C -rhGH

0.24 15 1.6 151 1.4 3 28 60 105

0.99 87 15.1 1441 3.2 23 99 173 748

2 2 i2 ? 2 ? t 2

0.47 20 2.6 182 0.5 4 33 34 155

Group H +rhGH 9.87 231 185 2199 12.5 20 87 163 654

t t + 5 5 ? 2 -c 2

Group C -rhGH

2.41t 36t 20t 2067 3.7 2 29 25 97

2.84 168 77 1684 10.0 23 116 174 575

2 0.72 + 19 If: 22 t 108 t 1.5 %2 2 28 ? 29 5 36

Mean ? SEM; n = 10 each trauma group. GH, growth hormone; IGF-1, insulin-like growth factor-l; IGFBP-3, major binding protein of IGF-1; rhGH, recombinant human growth hormone. * Normal values from references 3 and 4, and J. Trauma 1955;39:295. t P 5 0.05 vs patients without rhGH (Group C).

lated to be 1.06 ? 0.06% REE in rhGH patients compared with 0.57 ? 0.12% REE (P = 0.05) in patients without rhGH. Although the energy cost of this cycling is a small fraction, its significant increase due to rhGH in association with a corresponding increase in total lipolysis rate emphasizes the importance of this cycle as an adaptive measure to regulate mobilization of fat as a fuel energy source in injury. DISCUSSION

The metabolic effects of rhGH were investigated in a group of highly catabolic and hypermetabolic trauma patients who received full maintenance parenteral nutrient intake ( 1.1 times measured REE calories and 250 mg N *kg-’ * d-‘) for 7 d in the early catabolic stage of injury. Nitrogen balance, an index

of net whole-body protein balance is significantly improved in rhGH patients. Stimulation of whole-body protein synthesis is primarily responsible for the better nitrogen retention in’injured patients given adjunctive rhGH. Similar effects are seen in patients receiving rhGH and TPN after gastrointestinal disease.34 An acute anabolic action of rhGH on the net balance of amino acids across the leg or forearm also appears to be mediated by a stimulation of muscle protein synthesis rather than a suppression of proteolysis.20X35The mechanism of rhGH-induced anabolism may include direct effects as well as indirect effects mediated by endogenous hormonal mediators. Many of the anabolic effects of growth hormone on body tissues are mediated via stimulation of endogenous IGF-1 synthesis, which may exert anabolic effects via an endocrine and/or autocrinel

TABLE IV. SELECTED PLASMA FREE AMINO ACID LEVELS (bmol/L) IN TRAUMA PATIENTS’“’ Trauma: Day 0 Amino Acids Valine Phenylalanine Alanine Arginine Glycine Glutamine Omithine Serine BCAA NEAA TAA

Normals 219 50 397 75 271 533 69 103 394 2143 3035

2 2 2 3 ” 31 + 7 5 25 ? 126 rfr 6 t 5 -+ 22 ? 76 2 108

Group H 246 69 200 61 137 314 48 72 474 1218 2091

? 38 ? 5* 2 25* t 8 ” 14” ? 21* of-4* 2 9* 2 78 ? 124* 2 188*

Trauma: Day 7 Group C

195 2 60 ? 199 5 43 t 96 + 279 ? 34 1 62 t 414 -c 987 ? 1713 2

25 8 33* 5* 18* 22* 5* 6* 49 99* 176*

Group H 251 5 14 115 jl 12* 398 ? 247 94 ? 5* 242 + 19 366 t 30* 102 2 4*t 102 ZY6t 435 2 18 1843 2 144 2956 + 258t

Mean ? SEM. (a) From Ref. 8; * P 5 0.05 vs normal; t P zz 0.05 vs group C for that day. BCAA, branched chain amino acids (ref. 3): NEAA, nonessential amino acids (ref. 14); TAA, total amino acids (ref. 22).

Group C 218 81 250 86 170 335 72 76 400 1403 2229

? 22 2 15* 2 32 2 12 -e 19* -c 31* -c 6 ? 8* + 49 * 147* 2 228*

GROWTH

HORMONE

SUPPLEMENTATION

IN TRAUMA

PATIENTS

783

TABLE V. DAILY URINARY EXCRETION PARAMETERS IN TRAUMA, PATIENTS Trauma: Day 0 Group H N Balance (mg N *kg-’ . d-‘) Urea (gN/d) Creatinine (hmol/d) 3-MHS (bmolld) 3-MHSKreatinine (molar ratio OHPR (bmol/d) Orotic acid (mg/d) Uric acid (mg/d)

-223 12.7 12.85 464 4.57 676 5.04 1260

lo-‘)

X

i + 2 t + + ? +

Trauma: Days 5. 6, and 7 (average) Group C

25t 2.5 2.16 87’i 0.97t 117t 0.76 33t

-206 14.9 15.85 547 3.45 706 6.20 1553

2 + 2 2 t 2 -t +

17t 2.5t 1.23 58 0.37 146t 0.64 136t

Group H -41 13.5 10.58 204 2.17 1034 5.15 749

2 + -t ? ? + t -c

Group C

18* 1.0* 0.77* 28* 0.22* 87 0.28 97

-131 21.2 13.99 431 3.10 1031 5.70 1070

-t -c t 2 + 2 2 -e

14 1.1 0.57 29 0.15 71 0.26 71

Mean ? SEM. 3-MHS: 3-methylhistidine; OHPR: hydroxyproline. * P c 0.05 vs Group C (no rhGH) patients for that day: t P 5 0.5 vs days 5, 6, and 7 (average).

paracrine mechanism. Although intravenous nutrition itself increased plasma IGF- 1 levels, adjunctive rhGH doubled the IGF-

1 levels. It is likely that enhanced IGF-1 synthesis contributes to the protein anabolic effects of rhGH. The improvement in plasma IGFBP-3, the major binding protein of IGF-1, indicates more bioavailability of free IGF-1 for anabolic responses. Insulin may also represent an additional anabolic mediator. Growth hormone-induced increases in plasma insulin concentrations might enhance nutrient delivery to injured tissue for wound repair. Stimulation of collagen synthesis by rhGH has been demonstrated in the healing of colonic anastomosis in rats,36 and in the donor site healing of infant37 and adult3’ bum patients. When collagen is broken down, the amino acid, OHPR is released and largely excreted unchanged. It has been shown that mobilization of collagen does occur during starvation and protein deficiency, or in other states of net protein catabolism.3g-4’ A higher excretion of OHPR may be a specific indicator of increased collagen content/mobilization. Normal subjects (unpublished data, rr = 14) excrete 202 % 56 pmol OHPR/d, which

is increased significantly to 690 t 31 in traumatized subjects. During 7 d of TPN, the excretory rate of OPHR is further increased to 1032 -+ 75 pmol/d irrespective of rhGH supplementation. This implies that complex collagen kinetics are not significantly altered due to rhGH in trauma victims with multiple injuries. Orotic acid is an anabolic intermediate in pyrimidine biosynthesis and originates from carbamyl phosphate, which is a common substrate for de novo synthesis of urea and the pyrimidines. The excretion of erotic acid reflects a balance between urea cycle capacity and the rate of pyrimidine utilization. A decline in plasma urea levels and urinary urea excretion is seen after rhGH administration in trauma patients indicating an overall protein anabolism and better utilization of the infused amino acids. These protein conserving properties of GH may primarily involve extra hepatic tissues, since no evidence for a specific hepatic protein conservation could be demonstrated.42 This is supported by the fact that rhGH has no effect on erotic acid excretion. The observed changes in erotic acid excretion

Correlation Between Daily Nitrogen Balance and JMHS Excretion tcm

Daily EMethyl Histidine Excretion During TPN - 0

T

r

0 -rhGH

??

0

No rhGH (Group C)

(Group C)

+thGH(Group H) Y-198-1.62x r = -0.66 p < O.Wl

- +rhGH (Group H)

MemfSEM

p-zLJ Ns

‘\

‘\ \ ??

t

*

t

t

t

??

P“‘Y----~----~~___~____~__._~ ‘.

Nltmgen Balance (mg N/kg/d)

FIG. 1. Daily 3-methylhistidine excretion in trauma patients with (Group H) or without (Group C) recombinant human growth hormone (rhGH) supplementation. TPN, total parenteral nutrition.

FIG. 2. Inverse relationship between daily 3-methylhistidine (3-MHS) excretion (Y, pmol/d) and daily nitrogen balance (X, mg N. kg-’ *d-’ ) in trauma patients with or without recombinant human growth hormone (rhGH). Y = 198 - 1.62X; r = -0.65; P 5 0.01.

GROWTH HORMONE SUPPLEMENTATION TABLE VI. CONTRIBUTION OF MUSCLE PROTEIN BREAKDOWN’“’ TO WHOLE-BODY PROTEIN CATABOLIC RATE Study II (day 7)

Study I (day 0) Group H (+rhGH) 3-MHS excretion (pmohd) Muscle Protein equivalent (gP*kg-’ *d-l) WBPB”” (gP *kg-’ *d-‘) Group C (-rhGH) 3-MHS excretion (pmoud) Muscle Protein equivalent (gP*kg-’ *d-l) WBPB’s’(gP* kg-’ -d-l) Muscle protein breakdown/ WBPB, (%) Group H Group C Normals’

204 rf: 28

466 5 87t

1.383 2 0.260t 3.876 -+ 0.3107

0.628 + 0.086 2.771 2 0.242

431 2 29*

547 ? 48

1.497 2 0.131 3.739 -c_0.21o’r

1.228 2 0.083* 2.580 i 0.254

36 2 5t 40 2 4 33 + 2t

23 ? 3 48 t 3* 67 2 3

Mean 2 SEM. (a) Muscle protein breakdown is calculated from 3-MHS excretion (4.2 pmol 3-MHS/g mixed muscle protein, ref. 28.). (b) WBPB, whole body protein breakdown rate; Refer to Table VII. (c) From ref. 29. * P 5 0.05vsGroup H (Study II); t P 5 0.05 vs Study II.

are not likely to be caused by any urea cycle enzyme deficits or activities. Supplementation of rhGH also has no effect on the decreased uric acid excretion. Urinary excretion of 3-MHS on a meat-free diet has been widely applied as an indirect determinant of muscle myofibrillar protein breakdown.27-29 Excretion of 3-MHS rose after major operation in TPN-fed control patients but not in rhGH-supplemented subjects.43 Similar results are reported in patients after major gastrointestinal surgery.44 Fasting trauma patients under

IN TRAUMA

PATIENTS

basal conditions excreted 506 -t 68 pmol3-MHS/d compared with a low value of 235 2 13 pmol/d in fasted normals,29 indicating accelerated muscle protein breakdown as a trauma response. Mean muscle protein breakdown as a percentage of whole-body protein breakdown in multiple-trauma patients is 38 ? 5% compared with 22-24%” in normal fed subjects on a meat-free diet, 33 ? 2%29 in starved normals, and 38% in trauma patients2’ Intravenous nutrition alone has increased this ratio to 48 t 3% in trauma patients compared with 67 + 3% in normal.29 However, in rhGH-supplemented patients it is significantly reduced to 23 + 3%. This indicates the sparing of muscle protein from breakdown and perhaps preferential increase in breakdown of visceral proteins by rhGH. Forearm efflux of 3MHS reflects the skeletal muscle protein breakdown in that tissue compartment and it is found to be less in rhGHtreated patientsa reflecting the results seen in the whole body. This may perhaps be due to a direct growth hormone effect. Administration of rhGH during intravenous nutritional support, in critically ill multiple-trauma patients, exaggerated the hyperglycemic response, in spite of a concurrent increase in plasma insulin levels. This hyperglycemia is found not to be due to enhanced hepatic glucose production or defective oxidation. Defective nonoxidative disposal (glycogen synthesis and storage/lipogenesis) may be one of the mechanisms responsible for the observed increase in hyperglycemia with rhGH. Glucose output might have been inhibited by hyperglycemia per se, independent of a rise in insulin levels. Other possibilities for the hyperglycemic, hyperinsulinemia during rhGH supplementation include impaired glucose transport activity into tissue cells as suggested for burn patients.45 A significant post-receptor defect may have contributed to the observed insulin resistance.46 Hyperinsulinemia may be due to stimulated secretion or decreased breakdown of insulin, The absence of any change in plasma C-peptide levels due to rhGH supplementation suggests that the endogenous insulin secretion is not stimulated in trauma patients. GH has both an early insulin-like and a later anti-insulin-like effect on the glucose metabolism?’ The early effect (very shortly after the hormone is given) appears to result from an increase in cellular permeability and is attained only with very large local concentrations of the hormone. The later anti-insulin-like effect may reside in peripheral tissues& and may also be insulinotropic.47 The inability to overcome the defect in glucose metabolism at high plasma insulin concentrations suggests that a significant postreceptor defect contributes to the observed insulin resistance.46

TABLE VII. WHOLE-BODY PROTEIN KINETICS”’ Trauma: Study I Normals: Study I Protein turnover (gP - kg-’ *d-‘) Protein breakdown (gP - kg-’ . d-‘) Protein synthesis (gP - kg-’ . d-‘) Protein synthesis efficiency (%) N balance”’ (mg N *mg-’ *d-‘)

2.43 2.43 1.84 76.3

? 5 t 2

0.10 0.10 0.08 3.3

-

Mean ? SEM. (a) From ref. 6; (b) Mean of last 3 d of TPN in Study II. *P 5 0.05 vs Group C without rhGH.

Normals: Study II 2.89 1.05 1.77 60.1

)_ 0.12 2 0.06 2 0.08 ? 2.6

-

Group H 3.88 3.88 2.51 64.2 -223

c_ 0.31 2 0.31 2 0.27 & 3.6 2 25

Trauma: Study II

Group C 3.74 3.74 2.46 65.3 -206

r t + ? ?

0.21 0.21 0.19 3.0 17

Group H 3.99 2.77 2.48 62.3 -41

? t ? ? -e

0.28 0.24 0.19* 2.2* 18*

Group C 4.03 2.58 1.78 48.0 -121

2 5 ? -e 2

0.16 0.24 0.18 2.8 19

GROWTH

HORMONE SUPPLEMENTATION

IN TRAUMA

785

PATIENTS

TABLE VIII. GLUCOSE KINETICS IN rhGH-TREATED TRAUMA PATIENTSa’ Study I (Basal)

Glucose plasma level (mg/dL) Glucose appearance (mg *kgg’ - mm’) Glucose clearance (ml -kg-’*min-‘) Glucose intake (mg *kg-’. mu-‘) Hepatic output (mg . kg-’ *mu-‘) Vcoz (mL/min) Plasma glucose oxidized (mg . kg-’ 1min-‘) Glucose uptake oxidized (%) Glucose recycling (%)

Study II (TPN)

Group H (+rhGH)

Group C (-rhGH)

138 t 9 3.45 2 0.23 2.53 + 0.16 0 3.45 + 0.23 223 -c 19 0.86 2 0.17 29.1 + 5.4 12 2 2

150 t 13 4.38 2 0.71 2.92 t 0.27 0 4.38 ? 0.71 287 + 44 1.00 lr 0.16 21.5 2 1.9 13 t 1

Group H (+rhGH) 256 6.57 2.73 4.64 1.84 303 1.84 34.2 10

Group C (-rhGH)

2 25 rt 0.64 ? 0.22 2 0.48 2 0.21 + 22 i 0.52 ? 6.2 -+ 2

202 Z 17* 7.67 2 0.81 3.84 2 0.26* 4.80 2 0.64 2.80 2 0.48 346 ? 6 2.71 2 0.55 36.5 + 6.3 82-2

Mean t SEM. rhGH, recombinant human growth hormone; TPN, total parenteral nutrition. (a) From ref. 9. * P < 0.05 when the % change from Study I was compared between Groups H and C.

Although it is well recognized in normal subjects that insulin stimulates overall in vivo glucose disposal and that glucose disposal increases with elevation in plasma glucose concentration as a result of mass action, glucose transport, instead of intracellular glucose metabolism, is rate-limiting for in vivo glucose uptake over a range of glucose flux rates induced by hyperglycemia and hyperinsulinemia.4* Infusion of rhGH in normal humans inhibited, not enhanced, glucose uptake by muscle.47 Similarly GH also inhibited the glucose transport activity in erythrocytes.49 GLUT4 is the major glucose transporter isoform expressed in skeletal muscle. A translocation of the GLUT-4 transporter from an intracellular pool to the cell surface was suggested for the similar molecular events underlying IGF-1 and insulin actions on glucose uptake in skeletal muscle.” A defect in such translocation of the

GLUT-4 transporter may perhaps be responsible for the inhibition of glucose transport activity during rhGH supplementation in trauma patients. Multiple post-receptor sites may also be involved and specific studies are needed to establish this effect. Our results in parenterally fed, severely injured trauma patients indicate enhanced rates of lipolysis, reesterification and net carbohydrate oxidation with decreased oxidation rates of fat and protein, resulting in no change in energy expenditure. Lipid mobilization and utilization are also accompanied by improvements in the utilization efficiency of protein flux, resulting in an increased nitrogen retention. Hyperglycemia after rhGH administration may be the starting event followed by increased reesterification for a better continuous supply of FFA for fuel utilization. It is possible that

TABLE IX. PARAMETERS OF ENERGY METABOLISM AND LIPID KINETICS IN TRAUMA PATIENTS@’ Study I (Basal) Group H (+rhGH)

RQ REE (kCa1 * kg-’ *d-‘) NFO (%REE) NC0 (%REE) NPO (%REE) TG hydrolysis (kCa1. kg-’ - d-‘) FFA reesterihcation (kCa1. kg-’ . d-‘)

0.74 28.5 72.3 4.7 23.0 22.4 1.6

_’0.02 2 1.6 -t 8.2 2 6.4 + 3.4 2 2.0 _c 2.0

Study II (TPN)

Group C (-rhGH) 0.72 32.3 72.1 7.4 19.5 33.8 8.5

_t 0.02 5 1.5 2 6.3 * 7.6 2 2.7 + 6.8 +- 1.9

Group H (+rhGH) 0.92 32.1 25.2 55.9 18.9 26.4 17.9

2 0.04* + 2.2* 2 5.2% _c 7.1* -c 2.0 2 2.4* _c 1.3*

Group C (-rhGH) 0.88 33.3 39.8 31.9 27.7 23.2 10.0

i ? + 2 2 2 2

0.04* 2.9 75* 10.1* 2.17 14.0*? 1.97

Mean -C SEM. (a) From ref. 7. * P c 0.05 compared with respective Group in Study I (paired t test); t P s 0.05 compared with Group H (analysis of variance, ANOVA). rhGH, recombinant human growth hormone; RQ, respiratory quotient; REE, resting energy expenditure; NFO, net fat oxidation; NCO, net carbohydrate oxidation; NPO, net protein oxidation; TG, triglyceride; FFA, free fatty acid.

786

GROWTH HORMONE SUPPLEMENTATION

the antilipolytic effects of insulin are blunted by GH. Simultaneous operation of increased lipolysis and TG synthetic process may allow the adipocytes to respond rapidly to changes in peripheral requirements of FFA, a metabolic fuel, or release of substrate for gluconeogenesis. Although nitrogen-sparing effects of rhGH have been demonstrated, the data is limited to analysis to indicate a clear clinical benefit in terms of reduced mortality, improved outcome, accelerated recovery or better wound healing. Ample evidence attests to the interaction of GH and IGF-1 with the immune system.51 Evaluation of the immune function and changes in immune parameters during rhGH therapy in trauma patients in the acute phase of injury may be beneficial. A combination therapy of GH and IGF-1 is substantially more anabolic

IN TRAUMA

PATIENTS

than either GH or IGF-1 alone5’ in normal volunteers, indicating that the synergistic effects of this combination therapy might be helpful in selected cases. Co-administration of GH and IGF-1 or GH and insulin may also compromise the potentially harmful side effect of GH-induced hyperglycemia in trauma patients. There may be maximal effects on protein kinetics, with GH increasing synthesis and IGF-1 or insulin reducing breakdown. It remains to be seen in the critically ill patient group. ACKNOWLEDGMENT

The generous gift of recombinant human growth hormone from Genetec, Inc., South San Francisco, California, is gratefully acknowledged.

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46. Bratush-Marrain PR, Smith D, DeFronzo RA. The effect of growth hormone on glucose metabolism and insulin secretion in man. J Clin Endocrinol Metab 1982;55:973 47. Merimee TJ, Rabin D. A survey of growth hormone secretion and actions. Metabolism 1973;22:1235 48. Fink RI, Wallace P, Brechtel G, Olefsky SM. Evidence that glucose transport is rate-limiting for in vivo glucose uptake. Metabolism 1992;41:897 49. Kanigur-Sultuybek G, Hatemi H, Guven M, Tasan E, Tezcan V. Effect of growth hormone in glucose transport and binding of insulin to receptors in erythrocytes. Acta Pediatr 1992;383(suppl):l06(A) 50. Lund S, Flyvbjerg A, Holman GD, et al. Comparative effects of IGF-1 and insulin in the glucose transporter system in rat muscle. Am J Physiol 1994;267:E461 51. Gelato MC. Growth hormone-insulin like growth factor- 1 and immune function. Trends in Endocrinology and Metabolism 1993;4: 106 52. Kupfer SR, Underwood LF, Baxter RC, Clemmons DR: Enhancement of the anabolic effects of growth hormone and insulin-like growth factor-l by use of both agents simultaneously. J Clin Invest 1993;91:391