THE OBSERVATION THAT exercise is one of the most

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1 /03/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 88(4): Printed in U.S.A. Copyright 2003 by The Endocrine Society doi: /jc Effect of Growth Hormone (GH) on Glycerol and Free Fatty Acid Metabolism during Exhaustive Exercise in GH-Deficient Adults J. GIBNEY, M. L. HEALY, M. STOLINSKI, S. B. BOWES, C. PENTECOST, L. BREEN, C. MCMILLAN, D. L. RUSSELL-JONES, P. H. SONKSEN, AND A. M. UMPLEBY Department of Diabetes and Endocrinology, Guys, Kings and St. Thomas School of Medicine, St. Thomas Hospital, London SE1 7EH, United Kingdom GH is an important regulator of fat metabolism at rest, but it is not known whether it regulates fat metabolism during exercise. To determine whether physiologic concentrations of GH influence fat metabolism during exercise, we randomized 16 GH-deficient adults, receiving long-term (mean duration, 5 yr) GH replacement, to either continue GH (n 8) or receive identical placebo (n 8) for a 3-month period. Metabolic studies, at rest, during and following exhaustive exercise were carried out at baseline and at the end of the 3 months. The rate of appearance of glycerol (glycerol Ra, an index of lipolysis) and free fatty acids (FFA, FFA Ra) and the rate of disappearance of FFA (FFA Rd) in the plasma were measured using infusions of 2 H 5 -glycerol and 1-13 C-palmitic acid. Changes in body composition were assessed using dual-energy x-ray absorptiometry scanning and anthropometric measurements. In the baseline studies, exercise resulted in an increase in plasma glycerol and FFA concentrations, glycerol Ra, FFA Ra, and FFA Rd (P < 0.001). Three months of GH withdrawal resulted in reductions in plasma glycerol and FFA, glycerol Ra, FFA Ra, and FFA Rd at rest (P < 0.05 vs. baseline) and during exercise (P < 0.05 vs. baseline and vs. GH treated). Lean body mass decreased after 3 months of GH withdrawal, but total body fat, trunk fat, waist circumference, and the sum of skinfold thicknesses increased after 3 months of GH withdrawal (P < 0.05 vs. baseline and vs. GH treated). Fasting insulin and homeostasis model assessment of insulin resistance decreased after 3 months of GH withdrawal (P < 0.05 vs. baseline and vs. GH treated). In summary, GH withdrawal for 3 months resulted in reductions in release of glycerol and FFA into the circulation and uptake of FFA into the tissues during intense exercise. These changes were accompanied by reduced lean body mass and increased total body and trunk fat. Further studies are required to determine whether reduced mobilization of fat during exercise contributes to reduced exercise capacity and increased body fat in GH-deficient adults. (J Clin Endocrinol Metab 88: , 2003) THE OBSERVATION THAT exercise is one of the most powerful stimuli for GH release suggests that it may have an important physiological role in this setting (1). Specifically, it has been hypothesized that GH may be important in facilitating mobilization of stored fat during or following exercise (2). Consistent with this is the finding that GH deficiency in adults is associated with reduced exercise tolerance (3), excessive fatigue, and increased central adiposity (4). Previous studies investigating the role of GH, during exercise, have failed to demonstrate any effect on fat metabolism (5, 6). Such studies, however, have either compared GH-deficient (GHD) subjects with normal subjects (6) or used octreotide to suppress endogenous GH release (5). The former studies have low statistical power because of their cross-sectional design and, furthermore, are limited by unavoidable differences in body composition and physical fitness between GHD and normal subjects. The latter study was designed to determine the effect of acute GH withdrawal and additionally may be confounded by other metabolic effects of octreotide (7). Studies in adipocytes isolated from GHD patients suggest that more longterm changes in GH status may be necessary to observe an effect on lipolysis (8, 9). We used GHD adults as a model to study the effects of GH on fat metabolism at rest and in response to maximal exercise. Studies were carried out in the same subjects on two occasions, once while receiving long-term physiological GH replacement and again after 3 months of withdrawal of GH. Measurement of the rate of appearance of glycerol and rates of appearance and disappearance of palmitic acid provided an estimate of whole-body rates of lipolysis and free fatty acid (FFA) release and uptake. Changes in body composition were assessed using dual-energy x-ray absorptiometry (DEXA) scanning and anthropometric measurements. Patients Patients and Methods Sixteen patients aged yr who were GHD and had been receiving GH for at least 1 yr were recruited. Diagnosis of GH deficiency was based on a peak serum GH response to insulin tolerance test less than 3 ng/ml during adequate hypoglycemia (10). All subjects were receiving stable pituitary hormone replacement for at least 1 yr before study commencement. There were no changes in replacement needs during the trial. All measures were made at baseline and after 3 months. Abbreviations: CV, Coefficient of variation; DEXA, dual-energy x-ray absorptiometry; FFA, free fatty acids; GCMS, gas chromatography mass spectrometry; GHD, GH deficient; Ra, rate of appearance; Rd, rate of disappearance; VO 2, oxygen consumption. Study design and treatment The study was double blind and placebo controlled. After completion of a baseline evaluation of eligibility, 16 patients were randomized by 1792

2 Gibney et al. Exercise and Fat Metabolism in GHD Adults J Clin Endocrinol Metab, April 2003, 88(4): the hospital pharmacy to receive either GH (Humatrope, Eli Lilly & Co., Indianapolis, IN; n 8) at their usual dose or an equal volume of visually identical placebo (n 8). Patients continued to be followed up as endocrinology outpatients throughout the study. Approval was given by the Ethics Committee of St. Thomas Hospital. All patients gave written informed consent. Metabolic protocol Subjects presented to the exercise laboratory at 0800 h after an overnight fast. In the days before the experiments, they ate their habitual diet and performed their habitual physical activities. From 24 h before the experiments, they refrained from vigorous physical activity. The subjects usual dose of GH or placebo was withheld on the night before the study and administered instead on arrival at the exercise laboratory (3 h before the commencement of exercise). This protocol was observed because it has previously been demonstrated that some of the metabolic effects of GH replacement can only be demonstrated while GH persists in the circulation (11). The subjects usual dose of glucocorticoid was administered at the same time. At 1000 h, an iv cannula was inserted into the antecubital fossa of one arm for isotope infusion and the contralateral dorsal hand vein, which was heated, for arterialized blood sampling (12). Baseline blood samples were taken before commencement of a primed (0.9 mol/kg) continuous infusion of 2 H 5 glycerol (0.06 mol/ kg min) and 1 13C palmitate (0.03 mol/kg min). Following 60 min of infusion, samples were taken at 5-min intervals to allow calculation of steady state kinetics. Following 80 min of infusion, subjects cycled on an exercise bicycle controlled by a Lode cycle ergometer (Lode Excalibur Sport, Grunningen, Holland). The exercise protocol was as: 7 min at 0.5 W/kg; 7 min at 1 W/kg; and 7 min at 1.5 W/kg. Workload was then increased by 20 W every 2 min until exhaustion. This protocol was designed on the basis of pilot studies in 21 GHD subjects and enabled this study population, with a wide variation in physical fitness, to exercise for at least 15 min while reaching exhaustion within 30 min. As previously described (36), the isotope infusion rate was doubled at the start of exercise to minimize changes in enrichment, which reduce the accuracy of calculating non-steady state kinetics. Blood samples were taken every 5 min for 40 min from the start of exercise, every 10 min for another 40 min, and every 20 min until the end of the experiment (120 min from the start of exercise). Indirect calorimetry Oxygen consumption (VO 2 ) and carbon dioxide production were measured with use of a CPX-D cardiopulmonary exercise testing system (Medical Graphics, Birmingham, UK). Expired gas was sampled continuously at the mouth. The concentration of dried gas was measured with analyzers accurate to 1% (Zirconia Oxide oxygen analyzer with a response time of 80 msec and infra-red carbon dioxide analyzer with response time of 130 msec). Gas volume was measured with a bidirectional differential pressure prevent pneumotach with accuracy of 3%. Analytic methods All isotopic enrichments were measured by gas chromatography mass spectrometry (GCMS) on a HP 5971A MSD (Agilent Technologies, Berkshire, UK). The isotopic enrichment of glycerol was determined using a method modified from Elia et al. (13). Glycerol was isolated from deproteinized plasma using ion exchange chromatography and the Tristrimethylsilyl derivative formed. GCMS analysis used electron impact ionization with selected ion monitoring of the ions at m/z 205 and 208. Glycerol concentration was measured using a commercially available colorimetric assay (Randox Laboratories Ltd, Co. Antrim, UK) on an automated analyzer. Palmitic acid enrichment and concentration was determined using a method described by Patterson et al. (14). The procedure involved the extraction of lipids from plasma and methylation with iodomethane (CH 3 I) to form fatty acid methyl esters. These were subsequently purified by solid-phase extraction chromatography. Enrichment was determined with selected ion monitoring of the ions at m/z 270 and 271. Concentrations were calculated by relating relative peak area response of the palmitate to that of the internal standard heptadecanoic acid (C17:0). Insulin was measured by double-antibody RIA [within-assay coefficient of variation (CV), 6%]. Insulin resistance was estimated in each subject using the homeostasis model assessment of insulin resistance with the following validated formula: fasting serum insulin ( U/ml) fasting plasma glucose (mmol/l)/22.5 (34). IGF-I was measured by double-antibody RIA after acid/ethanol extraction, using a commercially available reagent pack (Amersham, Arlington Heights, IL; withinassay CV, 5%). Body composition Patients had height (measured with a Harpenden stadiometer) and weight measured in a hospital gown. Circumferences were measured with a nondistensible flexible tape measure at the waist and hip. Skinfold thickness was measured using Holtain calipers (Holtain, Crymych, UK) at the biceps, triceps, subscapular, and suprailiac sites. Total body fat and lean body mass were measured using DEXA, which was performed using a whole-body scanner (QDR-2000, Hologic, Inc., Bedford, MA). DEXA can be used to estimate fat in specific anatomical regions (15), and in this study an abdominal region (trunk) was defined to assess abdominal fat mass (CV 2% for all measurements). Calculations An isotopic steady state was achieved during the last 20 min of the basal (preexercise) period, so Steele s equation for steady state kinetics was used (16). During and following exercise, non-steady state conditions were present and Steele s equation for non-steady state kinetics was used to determine glycerol and palmitate rate of appearance (Ra) and palmitate rate of disappearance (Rd) (16). The volume of distribution of glycerol was assumed to be 230 ml/kg and the volume of distribution of palmitate was assumed to be 40 ml/kg (17). The Ra FFA was calculated by dividing the Ra of palmitate by the fractional contribution of palmitate to the total FFA concentration, as determined by GCMS analysis. Values for Ra and Rd at peak exercise were taken as an average of the last two values recorded during exercise, and postexercise values were calculated as the average of values recorded at 20 and 30 min after exercise. Statistical analysis Treatment groups were uncoded only after a clean data file was established. Characteristics of the groups at baseline were compared using Student s unpaired t tests. Nonnormally distributed data were transformed logarithmically before statistical analysis. The effects of exercise and GH treatment or withdrawal on metabolic responses in each group were analyzed by ANOVA with repeated measures. Comparisons between changes from baseline in the GH-treated and GH-withdrawal groups were made using unpaired t tests. Two subjects (one in each group) did not take part in the 3-month exercise studies, and their results were not included in this part of the analysis. All comparisons were two tailed. A P value of less than 0.05 was taken as statistically significant. Results At baseline, patients randomized to continue GH (GHtreated group) were comparable with patients randomized to GH withdrawal (GH-withdrawal group) for age, gender, height, and body mass index (Table 1). Both groups had been receiving GH for a mean duration of 5 yr. Biochemical changes IGF-I decreased in the GH-withdrawal group (P vs. baseline and vs. GH treated) and did not change in the GH-treated group (Table 2). Fasting insulin and homeostasis model assessment of insulin resistance decreased after 3 months of GH withdrawal (P 0.05 vs. vs. baseline and vs. GH treated) and did not change in the GH-treated group

3 1794 J Clin Endocrinol Metab, April 2003, 88(4): Gibney et al. Exercise and Fat Metabolism in GHD Adults (Table 2). GH withdrawal resulted in decreased plasma glucose at rest and during exercise (see Table 4), compared with baseline (P 0.05). There were no changes from baseline observed in the GH-treated group (see Table 4). The duration of exercise did not change in either the GHtreated (22 2to22 2 ml/min) or GH-withdrawal (21 3to21 3 ml/min) groups. Body composition GH withdrawal resulted in reduced lean body mass and increased total body fat, trunk fat, and ratio of trunk to nontrunk fat (P 0.05 vs. baseline and vs. GH treated, Table 3). Waist, but not hip, circumference, and the sum of skinfold thicknesses all increased after GH withdrawal (P 0.05 vs. baseline and vs. GH treated, Table 3). There were no changes from baseline observed in the GH-treated group (Table 3). Aerobic performance There were no baseline differences in exercise characteristics between those randomized to continue GH and those randomized to GH withdrawal. There was a trend toward reduced VO 2 at peak exercise after GH withdrawal ( to ml/min, P 0.08 vs. baseline) but not in the GH-treated group ( to ml/min). TABLE 1. Baseline characteristics of the study subjects GH-treated group GH-withdrawal group Age (yr) 43 (25 68) 46 (25 66) Male/female 4/4 5/3 Duration of GH 70 (24 132) 72 (24 132) therapy (months) GH dose (mg/kg d) 0.6 ( ) 0.6 ( ) Height (cm) 163 ( ) 168 ( ) BMI (kg/m 2 ) 25.4 ( ) 26.7 ( ) All values are median (range). FFA kinetics There was no change in plasma glycerol or FFA in either group between 80 min before exercise and the beginning of exercise (Fig. 1). A physiologic and isotopic steady state was achieved in both groups at both time points during the 20 min before exercise. There were no baseline differences between the GH-treated and the GH-withdrawn group in any component of FFA kinetics before exercise. In the baseline studies in both groups, there was an increase in plasma levels of glycerol and FFA (Fig. 1) and an increase in glycerol Ra, FFA Ra, and FFA Rd during exercise (Fig. 2). Glycerol Ra, FFA Ra, and FFA Rd declined after exercise, reaching resting values by 15 min post exercise. The decline in plasma glycerol and FFA concentration was more delayed, reaching resting values by 30 and 40 min, respectively, after exercise (Fig. 1). After withdrawal of GH for 3 months, resting levels of plasma glycerol, plasma FFA (Fig. 1), glycerol Ra, FFA Ra, and FFA Rd (Fig. 2) were all lower than in the baseline studies (P 0.05 vs. baseline). There was no significant increase in any of these variables during exercise, resulting in values lower than during the baseline studies (P 0.05 vs. baseline and vs. GH treated, Figs. 1 and 2). The effect of GH withdrawal on glycerol Ra did not differ from the effect on FFA Ra. There were no changes observed in the group that continued on GH, compared with the baseline studies (Figs. 1 and 2). TABLE 2. Endocrine and metabolic variables at baseline and 3 months in subjects who were randomized to continue GH (GH-treated) or to discontinue GH (GH-withdrawal) GH-treated GH-withdrawal Baseline 3 months Baseline 3 months IGF-I (nmol/liter) a Free T 3 (pmol/liter) Free T 4 (pmol/liter) Fasting insulin ( U/ml) b HOMA IR b All values are means SEM. a P for the comparison of change from baseline with change from baseline in the GH-treated group. b P 0.05 for the comparison of change from baseline with change from baseline in the GH-treated group. TABLE 3. Body composition at baseline and 3 months in subjects who were randomized to continue GH (GH-treated) or to discontinue GH (GH-withdrawal) GH-treated GH-withdrawal Baseline 3 months Baseline 3 months Lean body mass (kg) a Fat mass (kg) a Trunk fat (kg) a Nontrunk fat (kg) Waist circumference (cm) a Hip circumference (cm) Sum of skinfold thicknesses (cm) a All values are means SEM. a P 0.05 for the comparison of change from baseline with change from baseline in the GH-treated group.

4 Gibney et al. Exercise and Fat Metabolism in GHD Adults J Clin Endocrinol Metab, April 2003, 88(4): TABLE 4. Plasma levels of glucose at peak exercise and 30 min after exercise in the GH-treated and GH-withdrawal groups GH-treated GH-withdrawal Baseline 3 months Baseline 3 months a Glucose (mmol/liter) Pre-exercise Peak exercise Post-exercise All values are means SEM. a Significantly different from baseline (P 0.05). FIG. 1. Plasma FFA and glycerol at rest and during and following exercise at baseline and 3 months after randomization to continue GH replacement (GH treated, left) or receive placebo (GH withdrawal, right)., Baseline studies;, 3-month studies. Discussion This double-blind, placebo-controlled study demonstrates for the first time that GH withdrawal, in GHD adults, influences fatty acid metabolism during intense exercise. Following 3 months of withdrawal of long-term GH replacement, there was a reduction in the release of both glycerol and FFA into the circulation and a reduction of tissue uptake of FFA at rest and during exercise. Predictable changes in body composition were seen after 3 months of GH withdrawal, with a reduction in lean body mass and an increase in total body and trunk fat. These data provide preliminary evidence that GH may have important effects on intermediary metabolism during exercise. Glycerol Ra provides an index of lipolysis because glycerol released during hydrolysis of adipose tissue cannot be reincorporated into triglycerides, the enzyme glycerol kinase being virtually absent in adipocytes (18). FFA Ra reflects the rate of release into the circulation of fatty acids. Unlike glycerol, fatty acids released during lipolysis can be reesterified to form new triglycerides, a process called triglyceride fatty acid cycling, and thus, FFA availability during exercise reflects not only changes in lipolysis but also changes in fatty acid reesterification within adipocytes. In the current study, GH withdrawal resulted in a similar reduction in glycerol Ra and FFA Ra during exercise, suggesting a reduction in both lipolysis and release of FFA into the circulation, without a major change in the rate of reesterification. This finding using stable isotopes was reflected in a reduction in plasma levels of glycerol and FFA. The rate of disappearance of FFA from the circulation was also reduced after GH withdrawal, which may partly reflect reduced availability of FFA, secondary to reduced FFA release into the circulation.

5 1796 J Clin Endocrinol Metab, April 2003, 88(4): Gibney et al. Exercise and Fat Metabolism in GHD Adults FIG. 2. Glycerol Ra, FFA Ra, and FFA Rd at rest and during and 30 min after exercise at baseline and 3 months after randomization to continue GH replacement (GH treated, left) or receive placebo (GH withdrawal, right). #, Significantly different from rest; *, significantly different from baseline studies; P 0.05;, significantly different from GH treated. Reduced fatty acid availability during exercise could contribute to reduced exercise capacity in GHD adults (3). GHD adults have reversibly reduced maximum oxygen consumption (3, 22) but also show functional abnormalities during submaximal exercise (23). Reduced maximal oxygen consumption at least partly reflects reduced muscle mass (3), but the effects of changes in fuel availability on exercise capacity have not been investigated. During exercise, FFA uptake is largely into skeletal muscle, the rate of uptake being dependent on FFA availability (24) and ultimately the rate of lipolysis. Experimental studies have demonstrated that a small increase in circulating FFA reduces glucose uptake and glycogen depletion, and increases fat oxidation, during exercise (25 28). Consistent with this, in the current study, FFA uptake from the circulation and plasma glucose were lower, both at rest and during exercise after GH withdrawal. Further studies are now needed to determine whether GH increases fat oxidation during exercise, as it does at rest (29). The observed effects of GH withdrawal may not all reflect a direct effect of GH but may also relate to changes in levels or effectiveness of other hormones. Reduced circulating insulin and reduced insulin resistance using homeostasis model assessment provide evidence for increased insulin sensitivity after GH withdrawal. Resistance to insulin includes resistance to the antilipolytic effect of insulin, and, therefore, increased insulin sensitivity might be expected to

6 Gibney et al. Exercise and Fat Metabolism in GHD Adults J Clin Endocrinol Metab, April 2003, 88(4): reduce lipolysis. However, further complexity is added by the recent observation that inhibition of lipolysis by acipimox abolishes the effect of GH to increase insulin resistance, suggesting that stimulation of lipolysis by GH might explain its effect on insulin sensitivity (35). Therefore, the relationship among GH, insulin, and FFA remains extremely complex and requires further investigation. Although, we studied subjects 3 h after their most recent GH injection, we consider it highly improbable that the effects observed during exercise represent a delayed effect of the last GH injection. First, there was no change in circulating FFA or glycerol during the 80 min before exercise. Second, an isotopic steady state was present before exercise. These findings suggest that GH stimulation of lipolysis had reached a plateau and are in keeping with in vitro and in vivo studies of the time course of GH-stimulated lipolysis (2, 33). Finally, the observation that all measures of fat metabolism declined to resting levels, within 30 min of cessation of exercise, is consistent with an acute effect of exercise rather than the more prolonged effect of GH stimulation. In summary, we have demonstrated that GH replacement, in GHD adults, influences fat metabolism during exhaustive exercise as well as at rest. GH withdrawal decreased the rate of appearance of glycerol and fatty acids in the circulation and decreased fatty acid uptake from the circulation. We propose that reduced fat turnover during exercise may contribute to changes in exercise tolerance and body composition in adult GH deficiency. 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