Short-Term Treatment with Low Doses of Recombinant Human GH ...

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The Journal of Clinical Endocrinology & Metabolism 87(7):3105–3109 Copyright © 2002 by The Endocrine Society

Short-Term Treatment with Low Doses of Recombinant Human GH Stimulates Lipolysis in Visceral Obese Men PAOLA LUCIDI, NATASCIA PARLANTI, FEDERICA PICCIONI, FAUSTO SANTEUSANIO, PIERPAOLO DE FEO

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Department of Internal Medicine, Section of Internal Medicine, Endocrine and Metabolic Sciences, University of Perugia, Perugia 06126, Italy This study was designed to explore whether low doses of recombinant human (rh)GH affect lipid, glucose, or protein metabolism in men with visceral obesity. Four different studies were performed in six, otherwise healthy, obese men (age, 42 ⴞ 3; body mass index, 33 ⴞ 1 kg/m2; waist circumference, 111 ⴞ 3 cm; mean ⴞ SEM). Lipid, glucose, and protein kinetics was estimated by infusing stable isotopes (glycerol, glucose, leucine) in the basal state and after 1 wk of treatment with sc bedtime injections of either placebo, 2.5 (GH2.5), or 3.3 (GH3.3) ␮g rhGH/kg body weight per day. When compared with baseline, placebo had no effect on lipid, glucose, or protein fluxes. In contrast, GH2.5 and GH3.3 increased lipolysis by approxi-

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ERUM GH CONCENTRATIONS are reduced in obese subjects due to blunted secretion (1, 2) and increased clearance (1, 3) of GH. In obesity, the frequency of GH bursts is maintained, but their magnitude is reduced (1, 2). The impairment in GH production is positively related to the amount of visceral fat (2, 4). Serum IGF-I levels, an integrated measurement of GH secretion, are inversely proportional to visceral fat mass but are apparently independent of sc fat mass (2, 4). Abdominal obesity might reduce GH secretion through inhibitory feedback exerted by increased concentrations of FFA (5) and/or insulin (6). Because GH increases lean body mass (therefore BMR) and decreases fat mass (especially visceral fat), reduced GH action in obesity leads to a vicious cycle worsening body composition. This hypothesis is indirectly supported by the evidence that untreated GH-deficient (GHD) adults have reduced lean body mass and increased visceral fat mass, which are normalized after recombinant human (rh)GH replacement therapy (7). In obese humans, a number of trials have demonstrated that treatment with pharmacological doses of GH [8.6 –50 ␮g/kg body weight (BW) per day] is effective in reducing fat mass (8 –11), especially visceral fat (9, 11), and in sustaining lean body mass (8 –11). However, treatment of visceral obesity with GH has been limited to experimental trials and is not authorized for current clinical practice, because no studies support the risk-benefit analysis. Pharmacological treatment with GH might worsen insulin resistance (12), commonly associated with visceral obesity, and augments serum Abbreviations: BAS, Basal state; BMI, body mass index; BW, body weight; FT3, free T3; FT4, free T4; GH2.5, 2.5 ␮g rhGH/kg body weight per day; GH3.3, 3.3 ␮g rhGH/kg body weight per day; GHD, GHdeficient; KIC, ␣-ketoisocaproic acid; PL, placebo; rh, recombinant human.

mately 25% (P < 0.04) without changing glucose and protein turnover rates. The two rhGH treatments increased within the normal range serum IGF-I (by ⬃30%; P < 0.01), whereas they augmented insulin secretion (P < 0.04) in a dose-dependent manner (GH2.5 by 19%, GH3.3 by 37%). C-peptide secretion was increased (P ⴝ 0.01) only by GH3.3 (by 28%). In conclusion, 1 wk of treatment with low doses of rhGH is sufficient to increase lipolysis in visceral obese men, but it does not modify glucose and protein turnover rates. The results of this study provide the rationale to design clinical trials using low doses of rhGH to attempt to reduce fat mass. (J Clin Endocrinol Metab 87: 3105–3109, 2002)

IGF-I that has been associated with an increased risk of developing several tumors (13, 14). At present, no trial has been performed using doses of rhGH enough to normalize serum GH concentrations of visceral obese subjects. The rationale behind this trial depends on the demonstration that low doses of rhGH are able to increase lipolysis in visceral obese humans. The present study was designed to test this hypothesis. The rates of whole body lipolysis, proteolysis, glucose appearance, and disposal have been compared in six visceral obese men after 1 wk of treatment with placebo, 2.5 (GH2.5), or 3.3 (GH3.3) ␮g rhGH/kg BW䡠d. Subjects and Methods Subjects After receiving the approval of the Ethical Committee of Umbria Region, we obtained informed written consent from six adult men (age 33–50 yr) whose physical characteristics are reported in Table 1. Five of six subjects were obese [body mass index (BMI) ⬎30 kg/m2], and one subject was overweight (BMI, 28.4 kg/m2); five subjects had visceral obesity (waist circumference ⬎102 cm), and one had a borderline waist circumference (101 cm). The volunteers, aside from obesity, were in good health as determined by medical history, physical examination, and laboratory evaluation.

Design of the study The subjects were studied on four different occasions at 7-d intervals. Until the completion of the entire study, all volunteers consumed a diet of 35 kcal䡠kg⫺1䡠d⫺1 containing 55, 30, and 15% carbohydrate, fat, and protein, respectively. After the first study, which was performed to measure lipid, glucose, and whole-body protein kinetics in the basal state (BAS), the volunteers were given, at bedtime, seven daily sc injections of three different interventions: placebo (vehicle), GH2.5, or GH3.3 (Humatrope, Eli Lilly & Co., Sesto, Florence, Italy). The study was double blind and the sequence of treatments was randomized. On the morning of the 8th, 15th, and 22nd d, i.e. 12 h after the 7th injection of

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Lucidi et al. • rhGH Therapy in Visceral Obesity

TABLE 1. Physical characteristics of the subjects

Age (yr) Weight (kg) Height (cm) BMI (kg/m2) Waist (cm) Hip (cm) Waist/hip

1

2

3

4

5

6

Mean

50 115 180 35.5 115 118 0.97

39 111 175 36.2 118 116 1.02

37 120 184 35.4 117 119 0.98

45 103.5 181 31.6 101 103 0.98

45 101 175 33 111 109 1.02

33 92 180 28.4 103 112 0.92

41.5 107.1 179.2 33.4 110.8 112.8 0.98

TABLE 2. Circulating concentrations of plasma glucose, plasma nonesterified FFA, serum insulin, and C-peptide in the BAS and after 1 wk of daily treatment with either PL, GH 2.5, or GH 3.3

Glucose (mmol/liter) FFA (␮Eq/liter) Insulin (pmol/liter) C-peptide (nmol/liter)

BAS

PL

GH2.5

GH3.3

4.6 ⫾ 0.2 471 ⫾ 29 104 ⫾ 18 2.7 ⫾ 0.3

4.6 ⫾ 0.1 435 ⫾ 44 112 ⫾ 18 2.9 ⫾ 0.3

4.6 ⫾ 0.1 430 ⫾ 44 133 ⫾ 17a 3.3 ⫾ 0.3

4.7 ⫾ 0.1 432 ⫾ 54 153 ⫾ 23a,b 3.7 ⫾ 0.5a

Data are expressed as mean ⫾ a P ⬍ 0.04 vs. PL. b P ⬍ 0.03 vs. GH2.5.

SEM.

either placebo, GH2.5, or GH3.3, lipid, glucose, and whole-body protein kinetics was estimated.

Assessment of lipid, glucose, and protein kinetics The patients were admitted to the Clinical Research Center of our department at approximately 0730 h, after an overnight fast. At approximately 0800 h, an 18-gauge plastic catheter needle was placed in an antecubital vein for the infusion of [1,1,2,3,3-D5]glycerol, [6,6D2]glucose, and l-[5,5,5-D3]leucine (purchased from Cambridge Isotopes Laboratories, Andover, MA) by three Harvard syringe pumps (Harvard Apparatus, Ealing, South Natick, MA) and saline (0.5 ml䡠min⫺1; Vial Me´ dical pump, Grenoble, France). A contralateral hand vein was cannulated in a retrograde fashion with a 19-gauge butterfly needle, and the hand was maintained at 65 C in a thermoregulated Plexiglas box for intermittent sampling of arterialized venous blood (15). At approximately 0900 h (0 min), a primed-constant iv infusion of [1,1,2,3,3-D5]glycerol (prime 6 ␮mol䡠kg; infusion rate, 0.2 ␮mol䡠kg⫺1䡠 min⫺1), [6,6-D2]glucose (prime 300 mg; infusion rate, 3.3 mg䡠min⫺1) and l-[5,5,5-D3]leucine (prime 1 mg䡠kg; infusion rate, 1 mg䡠kg⫺1䡠h⫺1) was started and continued for 5 h. Four milliliters of blood were collected at ⫺15, 0, 240, 270, and 300 min to measure the plasma concentrations and the enrichments of glycerol, glucose, leucine, and ␣-ketoisocaproic acid (KIC). Ten milliliters of blood were collected at 0, 240, 270, and 300 min to measure the concentrations of glucose, FFA, insulin, C-peptide, GH, IGF-I, ACTH, cortisol, free T3 (FT3), free T4 (FT3), TSH, and leptin.

Analytical methods The plasma concentrations of glucose were determined using a Beckman glucose analyzer (Beckman Coulter, Inc., Palo Alto, CA); those of FFA were determined using a colorimetric assay (Sclavo Diagnostici, Kite, Italy). The serum concentrations of insulin (Technogenetics, Milan, Italy), GH (Biodata, Ares Serono, Norwell, MA), and the plasma ACTH (DiaSorin, Inc., Stillwater, MN) concentrations were measured using commercial immunoradiometric assays. Serum IGF-I (acid alcohol extraction, Diagnostics Systems Laboratories, Inc., Webster, TX) was determined by RIA. With this method, the values of 10 healthy nonobese subjects (age 41 ⫾ 3 yr) were 243 ⫾ 35 ng/ml. C-peptide (Diagnostics Systems Laboratories, Inc.) and leptin (Mediagnost, Reutlingen, Germany) concentrations were determined using RIAs. The serum concentrations of TSH, FT3, FT4, and cortisol were determined by enhanced chemiluminescence using kits of Ortho-Clinical Diagnostics (Johnson & Johnson, New Brunswick, NJ). Leucine and KIC were extracted from plasma samples as previously described (16) after the addition of 50 ␮l/ml plasma of norleucine (160 ␮mol/ml) and 20 ␮/ml of ␣-ketocaproate (20 ␮mol/ml) as internal

standards. Glycerol was extracted from plasma as previously described (17) after the addition of [2-13C1]glycerol (0.1 ␮mol/ml solution) as internal standard (10 ␮l/ml of plasma). Glycerol enrichment and concentration were measured on its Tristrimethylsilyl derivative using gas chromatography mass spectrometry (GC 8000-Top, MS Voyager, ThermoQuest Italia, Rodano, Milan, Italy) in electron impact ionization mode monitoring the ions 205, 206, and 208 (18). Enrichments and concentrations of leucine and KIC were determined on their t-butyldimethylsilyl derivatives, enrichment of glucose on its penta-acetate (penta-O-acetyl-␤-d-glucopyranose) derivative. Enrichments of glucose, leucine, and KIC were measured using gas-chromatography mass spectrometry in electron impact ionization mode (GC HP 5890 II, MS HP 5972A, Hewlett-Packard Co., Palo Alto, CA), monitoring the ions 202/200 for glucose, 305/302 for leucine, and 304/301 for KIC (19).

Calculations The rates (␮mol䡠kg⫺1䡠min⫺1) of glycerol, glucose, leucine, and appearance were calculated by dividing the tracer infusion rate by the average enrichment of the tracee (KIC for leucine) measured over the last hour of each study (at 240, 270, and 300 min). Glucose clearance (ml䡠kg⫺1䡠min⫺1) was estimated by dividing glucose disposal rate by mean plasma glucose concentration at 240, 270, and 300 min.

Statistical analysis Statistical analyses were performed using repeated measure ANOVA for normally distributed data and Friedman’s test for data (C-peptide, leptin, FT3, and ACTH) which failed normality test. Where significant differences in mean responses were found, these were examined further using Tukey’s multiple comparison test. Data are presented as mean ⫾ sem; P values less than 0.05 were considered statistically significant. All analyses were run using Statistica 4.5 (StatSoft, Inc. 1993, Tulsa, OK).

Results

During the study, weight and waist circumference remained unchanged, and patients did not experience any adverse effect. Plasma concentrations of glucose, FFA, insulin, and C-peptide (Table 2)

Placebo (PL) treatment did not affect plasma glucose, FFA, serum insulin, and C-peptide concentrations. When compared with PL, GH2.5 and GH3.3 increased serum insulin (P ⬍ 0.04); the increase of insulin induced by GH3.3 was greater (P ⬍ 0.03) in comparison to that induced by GH2.5. In comparison to PL, GH3.3 significantly increased Cpeptide (P ⬍ 0.02), not GH2.5 (P ⫽ NS). Both treatments resulted in plasma glucose and FFA concentrations not different vs. PL. Hormone concentrations

There was no effect of treatments (P ⫽ NS) on plasma concentrations of ACTH and serum concentrations of corti-


sol, FT3, FT4, and TSH. The concentrations of ACTH, cortisol, FT3, FT4, and TSH were in the normal range. Also, serum leptin concentrations (nanograms per milliliter) were unaffected (P ⫽ 0.3) by treatments (BAS, 17.5 ⫾ 4.4; PL, 20.1 ⫾ 4.9; GH2.5, 21.7 ⫾ 4.3; GH3.3, 19.7 ⫾ 4.5). PL had no effect on serum GH (BAS, 0.91 ⫾ 0.27; PL, 1.06 ⫾ 0.20) and IGF-I (BAS, 209 ⫾ 25; PL, 215 ⫾ 22) concentrations. In comparison to PL, serum GH increased (P ⬍ 0.01) after GH2.5 (1.96 ⫾ 0.26) and GH3.3 (2.02 ⫾ 0.26), without differences between the two treatments. Also, serum IGF-I concentrations (nanograms per milliliter) were increased (P ⬍ 0.01) by GH2.5 (276 ⫾ 16) and GH3.3 (281 ⫾ 18), without differences between the two treatments and in comparison to those of 10 healthy nonobese volunteers (matched for age).

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Glucose and protein metabolism (Fig. 1)

The plasma concentrations of glucose, leucine, and KIC were unaffected by treatments. Glucose, leucine, and KIC enrichments were at steady-state over the last 60 min of the four studies. For these reasons, the rates of glucose appearance and disposal were the same. The rates of glucose appearance (BAS, 7.33 ⫾ 0.25; PL, 7.18 ⫾ 0.28; GH2.5, 7.27 ⫾ 0.31; and GH3.3, 7.53 ⫾ 0.45 ␮mol䡠kg⫺1䡠min⫺1), those of glucose clearance (BAS, 1.61 ⫾ 0.06; PL, 1.58 ⫾ 0.05; GH2.5, 1.53 ⫾ 0.06; and GH3.3, 1.59 ⫾ 0.09 ml䡠kg⫺1䡠min⫺1) and those of leucine appearance (BAS, 2.57 ⫾ 0.10; PL, 2.71 ⫾ 0.17; GH2.5, 2.64 ⫾ 0.12; and GH3.3, 2.65 ⫾ 0.13 ␮mol䡠kg⫺1䡠min⫺1) were unchanged by treatments. Discussion

Lipid metabolism (Fig. 1)

The plasma concentrations of glycerol were unaffected by treatments. The enrichments of plasma glycerol were at steady-state over the last 60 min (used to estimate substrate kinetics) of the four studies. There was no difference in the rate of glycerol appearance (␮mol䡠kg⫺1䡠min⫺1) between basal (3.15 ⫾ 0.35) and PL (2.97 ⫾ 0.22) studies. The rate of glycerol appearance significantly increased vs. PL after either GH2.5 (4.12 ⫾ 0.53; P ⬍ 0.04) or GH3.3 (3.85 ⫾ 0.44; P ⬍ 0.01), without differences between these two studies.

FIG. 1. Rates of glycerol appearance, leucine appearance, glucose appearance, and clearance in six visceral obese men in the BAS and after 1 wk of daily treatment with either PL, GH2.5, or GH3.3; data are expressed as mean ⫾ SEM. Ra, Rate of appearance. *, P ⬍ 0.04 vs. PL.

The results of this study demonstrate that 1 wk of treatment with GH2.5 increases by approximately 25% the rate of whole-body lipolysis without affecting glucose and protein kinetics. The rate of lipolysis was similarly stimulated by 1 wk of treatment with GH3.3. Also, this dose of rhGH did not change glucose and protein fluxes but increased serum Cpeptide and insulin concentrations by approximately 30 – 40%. The mechanisms through which GH activates lipolysis have been investigated in isolated hepatocytes and in adipose tissue of mice (20). After GH exposure, lipolysis can increase up to 3-fold (20), and it has been shown that the effect, in adipose tissue of mice, is mediated by signal transducer and activator of transcription 5 (21). The lipolytic effect of GH is direct, not mediated by IGF-I that has no receptors on mature adipocytes (22). Our estimates on the effects of rhGH on in vivo lipolysis are based on the dilution into the systemic circulation of labeled, exogenous glycerol by endogenous-released glycerol. It is possible that the measured rhGH effect of approximately 25% increase has been underestimated by our methodological approach. In fact, GH stimulates lipolysis especially in visceral adipose tissue (20), and it is likely that, because of first-pass liver metabolism, part of glycerol released by abdominal fat never did reach the systemic circulation. Thus, it is not possible to rule out that in our study rhGH treatment increased more than 25% visceral lipolysis. After rhGH treatment, the rate of glycerol appearance increased, but circulating FFA and glycerol concentrations did not significantly change. This result stresses the importance of measuring substrate kinetics instead of their circulating concentrations. In fact, the use of FFA and glycerol might have been accelerated by concomitant GH activation of FFA oxidation (20) and by GH-induced hyperinsulinemia that promotes triglyceride reesterification (23). In the BAS, our obese subjects had a clear insulin resistance with serum insulin concentrations 2-fold higher in comparison with those measured in healthy nonobese subjects in our laboratory (24). Accordingly, the rates of glucose fluxes (appearance and disposal) were almost half of those measured in our previous studies in healthy nonobese subjects (24). This means that obese subjects, despite very low rates of peripheral glucose disposal, maintained normoglycemia because of compensatory hyperinsulinemia that reduced hepatic glucose production (25). The administration of rhGH

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did not change the rate of glucose appearance but augmented, in a dose-dependent manner, insulin production. Increased insulin production was sufficient to counteract the effects of rhGH on glucose metabolism, not those on lipolysis. Thus, in visceral obese subjects, in contrast to glucose metabolism, lipolysis is more sensitive to the catabolic effects of GH than to the anabolic effects of insulin. The rates of leucine appearance of the visceral obese subjects were similar to those measured in our laboratory in healthy nonobese subjects (26) and were not affected by GH-induced hyperinsulinemia. In a previous study performed in GHD adults, we demonstrated that 1 wk of treatment with GH3.3 did not change the rate of leucine appearance, but increased protein synthesis because of reduced leucine oxidation (27). In this study, leucine oxidation was not determined; thus no conclusion can be drawn on possible positive effects of rhGH on protein synthesis. The fact that hyperinsulinemia was associated with normal, and not reduced, proteolytic rates suggests that visceral obese subjects develop a partial resistance to insulin action, as observed in type 2 diabetic patients (28). The differential threshold of rhGH action on lipid and glucose metabolism can be conveniently used to reduce fat mass without increasing plasma glucose concentrations. In this regard, it is important to underline that the rate of glycerol appearance increased to the same extent after the treatment with GH2.5 or GH3.3. The lower rhGH dose offers the advantage to induce less insulin resistance as documented by lower serum insulin and C-peptide concentrations. This dose of rhGH is closer to a physiological correction of the reduced GH production than to a pharmacological treatment. In fact, GH2.5 increased within the normal range serum GH and IGF-I concentrations. These low doses of rhGH have never been tested in clinical trials designed to evaluate the efficacy of GH treatment in obese humans (8 –11). The amount of rhGH used in these studies ranged between 8.6 (11) and 50 ␮g/kg BW䡠d (8). These doses induced significant fat loss but increased above the normal values of IGF-I concentrations and were associated with several side effects (8 –11). Paradoxically, when very large doses of rhGH have been used, the lipolytic effect of rhGH, assessed measuring serum FFA concentrations, was offset by marked hyperinsulinemia (29). The present demonstration that GH2.5 increases by approximately 25% whole body lipolysis warrants future long-term clinical trials with this dose. Such a near-physiological replacement of rhGH could avoid undesired IGF-I increase and the side effects of the treatment. Keeping serum IGF-I in the normal range is an important safety issue due to the mitogenic activity of IGF-I. Recent studies have found that IGF-I levels correlate with risk of prostate cancer in men, premenopausal breast cancer in women, lung cancer in men and women, and colorectal cancer in men and women (14). In a long-term trial, the mild insulin resistance observed after 1 wk with GH2.5 might be either worse or a transient side effect. The latter possibility will apply only if the lipolytic effect of rhGH persisted. In this case, long-term treatment is expected through the reduction of visceral fat to augment insulin sensitivity as demonstrated by Johannsson et al. (9) after 9 months of treatment of visceral obese subjects with 9.5 ␮g rhGH/kg BW䡠d.


In a study performed in GHD adults, we have observed a 21% increase of lipolysis after 1 wk of treatment with GH3.3 (27). The present results show that visceral obese subjects have a sensitivity to the lipolytic effects of rhGH at least similar to that of GHD adults. Clinical trials in GHD adults have clearly established that rhGH treatment in replacement doses reduces abdominal fat (7). It is likely that a long-term treatment with doses of rhGH of 2.5 ␮g/kg BW䡠d (or even lower) offers the potential for safely promoting lipolysis in visceral obese humans. The results of this study provide a scientific basis to design future clinical trials using this low dose of rhGH. Acknowledgments We are indebted to Romeo Pippi, Vania Cesarini, Francesco Cimarelli, Giampiero Cipiciani, Dr. Debora Mughetti, and Dr. Annalisa Brozzetti for their skillful technical assistance. We thank Dr. Domenico Valle of Eli Lilly & Co. of Italy for his helpful comments. Received November 28, 2001. Accepted March 11, 2002. Address all correspondence and requests for reprints to: Prof. Pierpaolo De Feo, Department of Internal Medicine, Section Internal Medicine, Endocrine, and Metabolic Sciences, Via Dal Pozzo, 06126 Perugia (I), Italy. E-mail: [email protected]. This study was supported by Grant 9906153187 from Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (Rome, Italy).

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Erratum Dr. A. O. Brinkmann was inadvertently omitted as an author of the article “Genotype versus phenotype in families with androgen insensitivity syndrome” by A. L. M. Boehmer, H. Bru¨ ggenwirth, C. Van Assendelft, B. J. Otten, M. C. T. Verleun-Mooijman, M. F. Niermeijer, H. G. Brunner, C. W. Rouwe´ , J. J. Waelkens, W. Oostdijk, W. J. Kleijer, T. H. Van Der Kwast, M. A. De Vroede, and S. L. S. Drop (The Journal of Clinical Endocrinology & Metabolism 86:4151– 4160). The correct listing of authors is as follows: A. L. M. Boehmer, A. O. Brinkmann, H. Bru¨ ggenwirth, C. Van Assendelft, B. J. Otten, M. C. T. Verleun-Mooijman, M. F. Niermeijer, H. G. Brunner, C. W. Rouwe´ , J. J. Waelkens, W. Oostdijk, W. J. Kleijer, T. H. Van Der Kwast, M. A. De Vroede, and S. L. S. Drop. The printer regrets the error.