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The Journal of Clinical Endocrinology and Metabolism logoLink to The Journal of Clinical Endocrinology and Metabolism
. 2009 Oct 9;94(11):4524–4532. doi: 10.1210/jc.2009-0381

Impact of Growth Hormone Receptor Blockade on Substrate Metabolism during Fasting in Healthy Subjects

Louise Moller 1, Helene Norrelund 1, Niels Jessen 1, Allan Flyvbjerg 1, Steen B Pedersen 1, Bruce D Gaylinn 1, Jianhua Liu 1, Michael O Thorner 1, Niels Moller 1, Jens Otto Lunde Jorgensen 1
PMCID: PMC2775657  PMID: 19820031

Abstract

Context: Experimental studies in GH-deficient patients and in healthy subjects receiving somatostatin-infusion suggest that GH is an important regulator of substrate metabolism during fasting. These models may not adequately reflect the selective effects of GH, and GH receptor (GHR) blockade offers a new model to define the metabolic role of GH.

Objective: The aim of this study was to investigate the impact of GHR blockade on substrate metabolism and insulin sensitivity during fasting.

Design: We conducted a randomized, placebo-controlled, crossover study in 10 healthy young men.

Intervention: After 36 h of fasting with saline or pegvisomant (GHR blockade), the subjects were studied during a 4-h basal period and 2.5-h hyperinsulinemic euglycemic clamp.

Main Outcome: We measured whole-body and forearm glucose, lipid, and protein metabolism, peripheral insulin sensitivity, and acyl and desacyl ghrelin.

Results: GHR blockade significantly suppressed circulating free fatty acids (1226 ± 83 vs. 1074 ± 65 μmol/liter; P = 0.03) and ketone bodies (3080 ± 271 vs. 2015 ± 235 μmol/liter; P ≤ 0.01), as well as forearm uptake of free fatty acids (0.341 ± 0.150 vs. 0.004 ± 0.119 μmol/100 ml · min; P < 0.01) and lipid oxidation (1.3 ± 0.1 vs. 1.2 ± 0.1 mg/kg · min; P = 0.03) in the basal period. By contrast, IGF-I levels in either serum or peripheral tissues were not impacted by GHR blockade, and protein metabolism was also unaffected. Basal glucose levels were elevated by GHR blockade, but insulin sensitivity was similar; this was associated with an increased acyl/desacyl ghrelin ratio.

Conclusion: GHR blockade, without changes in circulating or tissue IGF-I levels, selectively suppresses lipid mobilization and oxidation after short-term fasting. This supports the notion that stimulation of lipolysis is a primary and important effect of GH.

GH receptor blockade during fasting in healthy subjects suppresses lipid metabolism without a change in insulin sensitivity or protein metabolism.

GH is not only essential for promoting longitudinal growth and somatic maturation in childhood; it is also an important regulator of substrate metabolism. A central and early recognized feature of GH is stimulation of lipolysis and lipid oxidation (1,2). The lipolytic effect is causally linked to a concomitant impairment of hepatic and peripheral insulin sensitivity (3,4,5,6,7). In conditions of supraphysiological GH levels, clinically significant insulin resistance and glucose intolerance occurs (8,9). We have, however, also observed that the fasting-induced increase in GH levels, which amplifies lipolysis and also suppresses peripheral glucose uptake, constitutes a favorable protein-sparing adaptation (10,11,12). The data were obtained in GH-deficient patients studied with and without GH administration and in healthy subjects during a somatostatin infusion in combination with exogenous GH or saline. These models have shortcomings. First, it is difficult to emulate the increase in endogenous GH secretion. Second, GH-deficient patients may have additional pituitary deficits or comorbidity that could impact the effects of GH. Third, infusion of somatostatin together with insulin and glucagon may modulate the hepatic and peripheral actions of GH (13,14,15).

Pegvisomant is a specific GH antagonist that binds to the GH receptor (GHR) in competition with GH and blocks GHR signaling (16). In addition to being a licensed drug in the treatment of acromegaly (17), pegvisomant also provides a potential means to study the isolated actions of GH in healthy subjects.

In the present study, we evaluated the impact of pegvisomant-mediated GH blockade on substrate metabolism and insulin sensitivity after 36 h of fasting in healthy subjects. The methods included infusion of isotopes to assess protein turnover rates, indirect calorimetry to measure energy expenditure and oxidation rates, assessment of forearm substrate balances, and measurement of GHR-related signaling pathways and proteins in muscle and fat biopsies. In addition, acyl and desacyl ghrelin levels were measured with a novel and well-validated highly specific two-site sandwich assay, because it has been reported that ghrelin levels change with fasting (18) and may directly influence metabolism (19,20). The dose of pegvisomant was selected to reduce GHR activation without a major suppression of IGF-I levels (21).

Subjects and Methods

Subjects and experimental design

The study was approved by the Regional Committee on Biomedical Research Ethics and conducted according to the Declaration of Helsinki (2000) of the World Medical Association. The nature and potential risks were explained before participants gave written informed consent.

Ten healthy men were studied (age, 24.3 ± 0.6 yr; body mass index, 23.1 ± 0.4 kg/m2). Exclusion criteria included: family history of type 2 diabetes, use of medications, and alcohol consumption above 252 g ethanol per week.

In this randomized, single-blinded, placebo-controlled, crossover study, each subject was examined twice after 36 h of fasting with the administration of saline and with GHR blockade by pegvisomant (15 mg; Pfizer ApS, Ballerup, Denmark). We chose 15 mg of pegvisomant (equivalent to 0.20 ± 0.00 mg/kg in our study population) in an attempt to suppress GH actions without inducing major changes in IGF-I levels (21).

The study protocol is outlined in Fig. 1. The study periods were separated by a minimum of 6 wk. The subjects were instructed not to deviate from their normal level of activity, to abstain from alcohol, and to consume a diet complying with the national recommendations (maximum 30% of the energy from fat, 50–60% from carbohydrates, and 10–20% from protein) 3 d before each study period. Both saline and pegvisomant was injected sc in the abdomen at 2000 h to achieve a peak concentration and effect of GHR blockade during the subsequent metabolic study. During the fasting period, subjects were allowed to drink tap or mineral water and to perform normal ambulatory activities, excluding any kind of exercise. At 0700 h (after 35 h of fasting) the subjects were placed in a quiet, thermoneutral room. One iv cannula was placed in an antecubital vein for infusion. A second cannula was inserted retrogradely into a deep antecubital vein for deep venous sampling, and for arterial blood sampling a third cannula was inserted contralaterally in a dorsal hand vein and the hand was placed in a 65 C heated box for arterialization of the blood.

Figure 1.

Figure 1

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Study protocol after 36 h of fasting with placebo or GHR blockade. The level of pegvisomant throughout the GHR study day (mean ± se). See Subjects and Methods for further details.

Tracers: L-[15N]phenylalanine, L-[2H4]tyrosine, L-[15N]tyrosine, and [13C]urea

After blood sampling at t = 0, priming doses of L-[15N]phenylalanine (0.7 mg/kg), L-[2H4]tyrosine (0.5 mg/kg), L-[15N]tyrosine (0.3 mg/kg), and [13C]urea (390.6 mg) (Cambridge Isotope Laboratories, Inc., Andover, MA) were given and were followed by continuous infusion of L-[15N]phenylalanine (0.7 mg/kg · h), L-[2H4]tyrosine (0.5 mg/kg/h), and [13C]urea (42 mg/h) for 4 h.

Phenylalanine, tyrosine, and urea flux (Qu) was calculated as: (Qu) = I[Ei/Ep) − 1] in which I is the rate of tracer infusion (μmol · kg−1 · h−1) and Ei and Ep are enrichment of the tracer infused and plasma enrichment of the tracer at isotopic plateau, respectively (22). For calculation of the rate of phenylalanine conversion to tyrosine and incorporation into protein, the equations of Thompson et al. (23) were used. Regional phenylalanine kinetics, e.g. rate of protein synthesis (phenylalanine rate of disappearance) and protein breakdown (phenylalanine rate of appearance), were calculated as described by Nair et al. (24) and Copeland and Nair (25). Phenylalanine balance (PheBal) was calculated as: PheBal = (PheA − PheV) × F, where PheA and PheV are phenylalanine concentrations in arteries and veins, and F is blood flow. Urea nitrogen secretion rate (UNSR) was estimated as urinary excretion rate, corrected for accumulation in total body water and fractional intestinal loss (26).

Indirect calorimetry

The respiratory quotient and resting energy expenditure (REE) were estimated by indirect calorimetry, performed during the last 30 min of the basal and clamp period. Mean values of the last 25 min were used for calculations. Lipid and glucose oxidation were estimated after correction for protein oxidation, which were calculated on the basis of urea nitrogen excretion (27). Energy production (kilocalories per day) from lipid oxidation was calculated as: [(lipid oxidation × weight in kilograms)/1440 min/d] × 1000 and multiplied by the caloric density (9.1 kcal/g).

Forearm metabolism

Total forearm blood flow was measured by venous occlusion plethysmography before each deep venous sample. Metabolite uptake was calculated by multiplying the blood flow and the arteriovenous metabolite difference. Mean metabolite uptake was estimated over the last 30 min of the basal and clamp period.

Biopsies

At t = 60 min, muscle tissue from musculus vastus lateralis was obtained by a Bergström biopsy needle. The tissue was cleaned from blood and snap-frozen in liquid nitrogen. Subcutaneous fat tissue from the abdomen was obtained by liposuction at t = 240 min; it was cleaned from blood and frozen in liquid nitrogen.

Real time RT-PCR

IGF-I and SOCS-3 were analyzed as described previously (28). In short, the biopsies were homogenized using a polytron in the presence of TRIzol reagent (Invitrogen, Carlsbad, CA), and RNA was isolated as described in the manual from Invitrogen. cDNA synthesis was performed using the TaqMan kit N808-0234 (Perkin-Elmer, Boston, MA). The PCR was performed using SYBR Green Master mix (Applied Biosystems, Foster City, CA) with primers as described previously (28). Sufficient amounts of muscle and adipose tissue were available from nine and 10 subjects, respectively.

Determination of adipose tissue and muscle IGF-I content

IGF-I extraction from adipose tissue and muscle was performed as previously described (29). Briefly, 50 mg of tissue was homogenized on ice in 1 m acetic acid (500 μl) using a Retsch Mixermill 301 (Retsch GmbH & Co., Haan, Germany). The homogenate was extracted twice, and after lyophilization samples were redissolved in 500 μl of 40 mm phosphate buffer (pH 8.0). The tissue extracts were kept at −80 C until IGF-I assay was performed. Tissue IGF-I content in the homogenate was determined with a noncompetitive time-resolved immunofluorometric assay (30). Sufficient amounts of muscle and adipose tissue were only available from seven and six subjects, respectively.

Hyperinsulinemic euglycemic clamp

Insulin (0.6 mU/kg · min) (Actrapid; Novo Nordisk A/S, Bagsvaerd, Denmark) was infused from t = 240 to 390 min, and plasma glucose was clamped at 5 mmol/liter by adjusting the iv infusion rate of 20% glucose according to plasma glucose measurements every 5–10 min. Insulin sensitivity was estimated by the level of glucose infusion rate and expressed as insulin stimulated glucose uptake (M-value).

Two participants only completed the basal period because they developed a prolonged vasovagal episode in relation to micturition before the clamp.

Blood analysis

Plasma glucose was measured in duplicate immediately after sampling on Beckman Glucoanalyzers (Beckman Instruments, Palo Alto, CA). Serum samples were frozen immediately and stored at −20 C. GH and pegvisomant were analyzed as previously described (31). Insulin, cortisol, and total IGF-I were analyzed using time-resolved immunofluorometric assays (AutoDELFIA; PerkinElmer, Wallac, Turku, Finland). The normal range (0700 to 0900 h) for plasma cortisol was 201–681 nmol/liter. C-peptide was measured by ELISA (DakoCytomation, Cambridgeshire, UK), and free fatty acids (FFA) were analyzed by a commercial kit (Wako Chemicals, Neuss, Germany). Glycerol, acetoacetate, and 3-hydroxybutyrates (3-OHB) were measured using a COBAS biocentrifugal analyzer with fluorometric attachment (Roche Diagnostics, Welwyn Garden City, UK). Urea was determined by a commercial method (Cobas Integra 800, Roche, Mannheim, Germany). Glucagon was analyzed using in-house RIA. IGF-I bioactivity and IGF binding protein 1 (IGFBP-1) were measured at t = 0 and t = 390 only; IGF-I bioactivity was analyzed by a kinase receptor activation assay (32) and IGFBP-1 by an in-house RIA. Acyl and desacyl ghrelin were measured using two well-validated highly sensitive and specific two-site sandwich assays previously described by Liu et al. (18). Plasma concentrations of amino acids were determined by an HPLC system (Bio-Tek Kontron, series 525 and 465, fluorescence detector SFM25; Kontron Instruments, Milan, Italy).

Statistical analysis

Student′s paired t test or Wilcoxon signed rank matched pairs test was used where appropriate after testing for normal distribution by Kolmogorov-Smirnov. Skewed data were log-transformed before applying relevant statistical tests and were presented as medians and ranges. To examine whether measurements changed during the study day, the results obtained at the placebo day were subtracted from the pegvisomant day, and the difference was tested against the hypothesis of difference = 0 using univariate ANOVA for repeated measures. If measurements did not change during the basal period, data are presented as means of measurements from t = 0 min to t = 240 min and referred to as “basal”; data obtained during the clamp period are presented as means of measurements from t = 370 to t = 390. A P value < 0.05 was considered significant. All calculations were carried out using SPSS version 15.0 for Windows (SPSS, Chicago, IL).

Results

Hormones

In the study with GHR blockade, the levels of pegvisomant (micrograms per liter) remained constant during the basal period (883 ± 124, range 520–1480; ANOVA P = 0.32) and were not affected by the clamp (937.8 ± 146.2; P = 0.35) (Fig. 1). As shown in Table 1, GHR blockade did not significantly change the serum levels of GH, total IGF-I, IGF-I bioactivity, or IGFBP-1. Although, at the end of the clamp the levels of IGF-I bioactivity and IGFBP-1 were slightly lower during GHR blockade compared with placebo, this did not reach statistical significance [IGF-I bioactivity (μg/liter), 0.18 ± 0.12 (placebo) vs. −0.02 ± 0.12 (GHR blockade), P = 0.17; IGFBP-1 (μg/liter), −186 ± 38 (placebo) vs. −179 ± 40 (GHR blockade), P = 0.80].

Table 1.

Circulating levels of hormones

Placebo GHR blockade P
Cortisol (nmol/liter)
 Basal 404.1 ± 44.0 388.1 ± 27.2 0.44
 Clamp 250.0 ± 17.3 249.1 ± 14.7 0.95
Glucagon (ng/liter)
 Basal 110.2 ± 15.2 116.9 ± 17.1 0.5
 Clamp 13.9 ± 4.9 25.3 ± 4.8 0.26
Insulin (pmol/liter)
 Basal 9.61 ± 1.36 10.95 ± 1.79 0.25
 Clamp 177.31 ± 6.33 170.94 ± 8.82 0.26
C-peptide (pmol/liter)
 Basal 184.9 ± 21.1 207.0 ± 36.1 0.31
 Clamp 261.2 ± 42.3 222.0 ± 40.0 0.06
GH (μg/liter)
 Basal 4.8 (3.6–8.8) 5.3 (2.1–11.4) 0.47
 Clamp 0.7 (0.4–1.9) 0.8 (0.3–1.8) 0.73
Total IGF-I (μg/liter)
 Basal 248 (158–271) 209 (138–268) 0.29
 Clamp 225 (155–323) 195 (160–280) 0.4
Bioactive IGF-I (μg/liter)
 t = 0 2.08 ± 0.12 1.99 ± 0.16 0.57
 Clamp 2.30 ± 0.23 1.89 ± 0.19 <0.01
IGFBP-1 (μg/liter)
 t = 0 233.2 ± 40.0 227.6 ± 37.8 0.86
 Clamp 62.6 ± 10.7 50.0 ± 8.0 0.03
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Data represent mean ± se or median (range). 

As illustrated in Fig. 2, GHR blockade elevated the acyl/desacyl ghrelin ratio in the basal period (P = 0.05) as well as in clamp period (P = 0.01). GHR blockade did not significantly affect the levels of acyl ghrelin (picograms/milliliter) in the basal period (P = 0.11), but in the clamp period acyl ghrelin was approximately 37% higher (P = 0.02) compared with saline. Circulating levels of desacyl ghrelin (picograms/milliliter) were comparable (basal P = 0.19; clamp P = 0.17). Compared with the levels in the basal period, the clamp did not significantly change the levels of either acyl ghrelin (GHR blockade, P = 0.79; saline, P = 0.87) or desacyl ghrelin (GHR blockade, P = 0.83; saline, P = 0.59) or the ratio (GHR blockade, P = 0.23; saline, P = 0.84). In the basal period, the ratio of acyl/desacyl ghrelin correlated positively with basal glucose levels in both studies (GHR blockade, r = 0.60, P = 0.01; saline, r = 0.54, P = 0.02).

Figure 2.

Figure 2

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The levels of desacyl ghrelin, acyl ghrelin, the ratio (acyl/desacyl) and glucose during the study day, after 36 h of fasting with saline (black) and GHR blockade (white). Initiation of the hyperinsulinemic euglycemic clamp is marked by the gray line. Data are presented as mean ± se and are compared during the basal and clamp period.

Whole body metabolism

Lipid metabolism (Fig. 3A)

Figure 3.

Figure 3

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Whole body circulating levels (A) and forearm uptake (B) of FFA and 3-OHB after 36 h of fasting with saline (black) and GHR blockade (white) in the basal period and at the end of a hyperinsulinemic euglycemic clamp. Data are presented as mean ± se.

GHR blockade significantly suppressed circulating FFA levels compared with placebo, with a mean reduction (μmol/l) of 164 ± 68 (11.1 ± 4.6%) in the basal period and 69 ± 16 (40.1 ± 7.6%) in the clamp period. The levels of glycerol were comparable [basal, 84.7 ± 4.3 (GHR blockade) vs. 86.6 ± 5.8 (placebo), P = 0.73; clamp, 27.1 ± 3.4 vs. 25.4 ± 1.5, P = 0.65].

GHR blockade in the fasting state significantly suppressed 3-OHB and acetoacetate levels (data not shown). The impact of the hyperinsulinemic euglycemic clamp on lipid intermediates, as assessed by δ values (clamp-basal), did not differ between the two studies.

Protein and glucose metabolism (Table 2)

Table 2.

Protein and glucose metabolism

Placebo GHR blockade P
Whole body metabolism
 Urea (mmol/liter)
  Basal 5.6 ± 0.3 5.9 ± 0.2 0.09
  Clamp 4.8 ± 0.2 5.3 ± 0.3 0.09
 Urea flux (μmol/kg · h)
  Basal 437.0 ± 28.0 451.0 ± 29.0 0.68
 UNSR (mmol/liter)
  Basal 24.3 ± 2.1 23.9 ± 2.2 0.84
  Clamp 12.9 ± 1.4 14.3 ± 2.5 0.71
 Phenylalanine arterial (mg/liter)
  Basal 7.75 ± 0.26 8.11 ± 0.30 0.02
 Phenylalanine flux (μmol/kg · min)
  Basal 41.9 ± 1.1 42.3 ± 1.7 0.65
 Tyrosine flux (μmol/kg · min)
  Basal 27.4 ± 1.3 28.8 ± 1.8 0.19
 Phenylalanine conversion to tyrosine (μmol/kg · min)
  Basal 2.5 (1.8–3.5) 2.4 (1.9–6.5) 0.86
 Phenylalanine incorporation into protein (μmol/kg · min)
  Basal 39.3 (35.2–48.4) 37.4 (33.2–50.3) 0.86
 Protein oxidation (mg/kg · min)
  Basal 1.07 (0.78–1.17) 1.04 (0.75–1.56) 0.96
  Clamp 1.2 (0.71–1.52) 1.2 (0.76–1.27) 0.87
 Glucose (mmol/liter)
  Basal 3.7 ± 0.1 3.9 ± 0.1 0.01
  Clamp 4.9 ± 0.04 4.9 ± 0.04 0.87
 M value (mg/kg · min)
  Clamp 2.4 (1.3–3.1) 2.4 (1.6–5.9) 0.40
 Glucose oxidation (mg/kg · min)
  Basal 0.27 ± 0.11 0.42 ± 0.10 0.18
  Clamp 0.57 ± 0.14 0.66 ± 0.12 0.57
Forearm metabolism
 Phenylalanine balance (μg/100 ml · min)
  Basal −2.28 ± 0.71 −0.57 ± 0.63 0.04
 Phenylalanine venous (mg/liter)
  Basal 8.16 ± 0.24 8.19 ± 0.18 0.89
 Phenylalanine Rd (mg/liter)
  Basal 1.78 ± 0.42 1.77 ± 0.49 0.99
 Phenylalanine Ra (mg/liter)
  Basal 4.06 ± 0.93 2.34 ± 0.95 0.13
 Amino acid uptake (μmol/100 ml/min)
  Basal −1.08 ± 0.43 −0.32 ± 0.59 0.39
  Clamp −0.97 ± 0.58 −0.40 ± 0.37 0.40
 Glucose uptake (mg/100 ml/min)
  Basal −0.03 (−0.26 to 0.03) −0.03 (−0.12 to 0.04) 0.96
  Clamp 0.04 ± 0.02 0.03 ± 0.02 0.67
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Data are presented as mean ± se or median (range). Ra, Rate of appearance; Rd, rate of disappearance. 

When comparing GHR blockade and placebo, levels of amino acids in plasma were similar (data not shown). In addition, protein turnover (estimated by phenylalanine and urea flux), protein synthesis (phenylalanine incorporation into protein), amino acid degradation (determined as the rate of phenylalanine conversion to tyrosine), and UNSR were comparable.

The plasma levels of glucose in the basal period were significantly higher during fasting with GHR blockade as compared with placebo (Fig. 2), but insulin-stimulated glucose uptake (M value) did not significantly differ.

Forearm metabolism

Lipid (Fig. 3)

Fasting with GHR blockade caused significant suppression of forearm uptake of FFA compared with placebo, with a mean reduction (μmol/100 ml/min) of 0.34 ± 0.12 [48 (−205 to 864) %] in the basal period and 0.14 ± 0.03 [152 (−2264 to 320) %] in the clamp period. Forearm uptake of 3-OHB and glycerol release (data not shown) were not significantly affected by GHR blockade.

Protein and glucose (Table 2)

Forearm muscle exhibited a net release of phenylalanine in both studies, but phenylalanine balance was significantly less negative during GHR blockade as compared with placebo, primarily due to higher arterial levels of phenylalanine during GHR blockade. However, comparable incorporation of phenylalanine into and breakdown of forearm muscle protein, represented by phenylalanine rate of disappearance and rate of appearance, respectively, were observed during fasting with GHR blockade and placebo, and the forearm release of amino acids was not significantly different (data not shown).

The insulin stimulation during the clamp increased glucose uptake to the same degree in both studies.

Indirect calorimetry (Fig. 4)

Figure 4.

Figure 4

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Energy metabolism after 36 h of fasting with saline (black) and GHR blockade (white) in the basal period and at the end of a hyperinsulinemic euglycemic clamp, as assessed by lipid oxidation and REE (energy gain from lipid oxidation is illustrated with gray lines; basal P = 0.02, clamp P = 0.28). Data are presented as mean ± se.

The oxidation rate of lipid was significantly lower during GHR blockade compared with placebo, and so was REE (kilocalories/day) and energy gain from lipid oxidation (kilocalories/day) with a mean reduction of 108 ± 50 (5.2 ± 2.6%) and 79 ± 27 (12.1 ± 3.9%), respectively, in the basal period and 99 ± 22 (4.6 ± 1.1%) and 55 ± 46 (3.7 ± 11.3 9%), respectively, in the clamp period. Glucose and protein oxidation were comparable in the two studies (Table 2).

Biopsies

When comparing biopsies obtained during GHR blockade and placebo, no significant differences were recorded in SOCS3 mRNA and IGF-I mRNA. The tissue IGF-I content was not significantly reduced with pegvisomant (muscle, GHR blockade, 3.0 ± 0.4; saline, 3.8 ± 0.7, P = 0.18; adipose tissue, GHR blockade, 0.7 ± 0.1; saline, 0.6 ± 0.1, P = 0.24).

Discussion

GH secretion is amplified during fasting, and experimental data suggest that this may constitute a favorable metabolic adaptation (33). Previous studies have primarily been performed in patients with GH deficiency or in healthy subjects during suppression of endogenous GH production with somatostatin (10,11,12,34). We conducted a randomized, crossover study in healthy subjects during fasting with isolated and specific blockade of the GHR to further elucidate the impact of GH. GHR blockade selectively suppressed mobilization and oxidation of lipid intermediates in terms of suppressed levels of circulating FFA and ketone bodies, decreased forearm FFA uptake, and reduced oxidation at the whole body level. This effect of GHR blockade occurred without significant changes in either glucose or protein metabolism.

The pegvisomant dose used in the present study was selected on the basis of a study conducted by Thorner et al. (21) in order not to completely block GH activity, but rather to eliminate the metabolic effects of a fasting-induced GH increase. Accordingly, we obtained steady-state levels of pegvisomant, which were lower than those observed during conventional treatment of acromegaly (31). In line with previous studies (31,35), we observed a relatively large interindividual variation in serum pegvisomant levels; the mechanism underlying this variation remains uncertain and merits future investigations in a larger number of subjects.

The mechanism whereby GH promotes mobilization of lipids is not fully understood, but our finding of reduced levels of circulating FFA during GHR blockade is in line with a previous report where discontinuation of GH replacement in GH deficiency during fasting was associated with an attenuated rate of lipolysis (34,36). In our study, GHR blockade also caused a lowering of circulating ketone body levels during fasting. Ketone body production is stimulated by glucagon and inhibited by insulin, but FFA levels per se also seem to be a determinant (12,37). In addition, GH has been proposed to stimulate ketogenesis independently of ambient FFA levels (38). Both insulin and glucagon levels were unaffected by GHR blockade in our study, which supports a direct ketogenic action of either FFA or GH. Likewise, we did not observe an impact of pegvisomant on circulating cortisol levels. It must, however, be emphasized that we only measured the concentrations of FFA and ketone bodies and not the turnover of these metabolites.

We found reduced REE during fasting with GHR blockade. GH is known to augment REE, but the mechanism remains uncertain (39). Attenuated lipid oxidation is a well-known feature of fasting without GH (11), and in our study the reduction in REE caused by GHR blockade was not only accompanied by decreased lipid oxidation, but approximately 73% of the reduction in REE could be explained by reduced energy gain from lipid oxidation. In agreement with suppressed lipid oxidation, we found decreased forearm uptake of FFA, and forearm FFA uptake has previously been shown to be related to circulating FFA levels (6,40,41). Our present data in relation to GH and REE do not allow a clear distinction between cause and effect, but there seems to be a very close link between the lipolytic and calorigenic effects of GH (6).

We have previously observed that in the postabsorptive state exogenous GH exposure increases the expression of SOCS3 and IGF-I genes in muscle and fat in healthy subjects (42). In the present study, relative GHR blockade did not affect the expression of these genes during fasting, although it should be remembered that muscle tissue samples were only obtained from nine subjects. Clearly, more studies are needed to delineate the effects of GHR blockade on the expression of target genes.

It has previously been convincingly demonstrated that the insulin antagonistic effect of GH during fasting is linked to its lipolytic properties (4,5,6,7). Our present observation that GHR blockade did not significantly impact peripheral insulin sensitivity despite distinct lowering of FFA levels and uptake in skeletal muscle was therefore unexpected. The dose of pegvisomant, and thus the approximately 11% reduction in FFA levels in response to the GHR blockade may, however, have been too subtle to impact insulin sensitivity, a suggestion that is supported by the fact that FFA affects glucose metabolism in a dose-dependent manner (41) and that low-dose GH administration apparently does not impact glucose and protein turnover in healthy obese men despite an approximately 25% increase in lipolysis (43). It should be mentioned that glucose clamp data were only achieved in eight subjects. Unexpectedly, we observed augmented basal plasma glucose levels during GHR blockade despite unaltered levels of insulin, cortisol, and glucagon.

The proportion of acyl to desacyl ghrelin has previously been suggested to determine the overall metabolic response to ghrelin (19,20), and acyl ghrelin has been demonstrated to increase glucose levels and to reduce insulin sensitivity (19,44). Therefore, the elevated acyl/desacyl ratio during GHR blockade, in addition to the positive correlation between the acyl/desacyl ratio and glucose, may offer an explanation for the elevation in basal glucose levels. It is also possible that the elevated ghrelin levels abrogated the expected increase in insulin sensitivity after GHR blockade. Clearly, more experimental studies are needed on the impact of fasting on ghrelin acylation and on the putative direct effects of ghrelin on substrate metabolism and insulin sensitivity to substantiate or refute our hypothesis.

GH is important for protein preservation during fasting (11), and these effects are mediated by IGF-I as well as FFA and ketone body levels. In a study by Norrelund et al. (12) where healthy young men were examined during fasting with GH administration and concomitant suppression of lipolysis with acipimox, muscle protein breakdown was increased by approximately 50% along with a reduction in ketone bodies and IGF-I levels. However, restoration of FFA levels did not completely reestablish protein metabolism or ketone body levels. Despite significant reduction in both circulating levels and forearm uptake of FFA, we found that GHR blockade had no impact on any indices of whole body or forearm protein metabolism, except for a less negative forearm phenylalanine balance. Our results are in line with a recent report from Sakharova et al. (34) who reported unaltered proteolysis despite suppressed lipolysis when GH secretion was blocked in the fasting state. The lack of effects on protein metabolism in our study may be explained by several factors. It is possible that the reduction in FFA was too moderate to influence protein metabolism and that a higher dose of pegvisomant or a longer observation period would have given a different result. Whether ghrelin may affect protein metabolism remains to be further studied.

In summary, we have demonstrated that administration of pegvisomant in the fasting state in a dose that does not decrease circulating or tissue IGF-I levels selectively suppresses lipolysis and lipid oxidation without impacting insulin sensitivity or protein metabolism. This observation supports the hypothesis that stimulation of lipolysis is a primary and fundamental effect of GH, as originally predicted by Raben and Hollenberg (1). The lack of effect of short-term GHR blockade on insulin sensitivity may suggest that the insulin antagonistic effects of GH require relatively high GH levels.

Acknowledgments

We owe great thanks to J. Frystyk for performing the analysis on total IGF-I, IGF-I bioactivity and IGFBP-1. We thank medical laboratory technicians K. N. Rasmussen, H. F. Petersen, and E. S. Hornemann for excellent technical assistance. We also thank The World Anti-Doping Agency for financial support.

Footnotes

This study was sponsored by an unrestricted research grant from Pfizer Inc.

ClinicalTrials.gov id: NCT00476879.

Disclosure Summary: L.M., H.N., N.J., A.F., S.B.P., B.D.G., J.L., and N.M. have nothing to declare. M.O.T. received a National Institutes of Health grant (DK076037). J.O.L.J. received a research grant from The World Anti-Doping Agency and honoraria and consulting fees from Pfizer.

First Published Online October 9, 2009

Abbreviations: FFA, Free fatty acid(s); GHR, GH receptor; IGFBP-1, IGF binding protein 1; 3-OHB, 3-hydroxybutyrate; REE, resting energy expenditure; UNSR, urea nitrogen secretion rate.

References

  1. Raben MS, Hollenberg CH 1959 Effect of growth hormone on plasma fatty acids. J Clin Invest 38:484–488 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Møller N, Jørgensen JO, Alberti KG, Flyvbjerg A, Schmitz O 1990 Short-term effects of growth hormone on fuel oxidation and regional substrate metabolism in normal man. J Clin Endocrinol Metab 70:1179–1186 [DOI] [PubMed] [Google Scholar]
  3. Rabinowitz D, Klassen GA, Zierler KL 1965 Effect of human growth hormone on muscle and adipose tissue metabolism in the forearm of man. J Clin Invest 44:51–61 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Nielsen S, Moller N, Christiansen JS, Jorgensen JO 2001 Pharmacological antilipolysis restores insulin sensitivity during growth hormone exposure. Diabetes 50:2301–2308 [DOI] [PubMed] [Google Scholar]
  5. Piatti PM, Monti LD, Caumo A, Conti M, Magni F, Galli-Kienle M, Fochesato E, Pizzini A, Baldi L, Valsecchi G, Pontiroli AE 1999 Mediation of the hepatic effects of growth hormone by its lipolytic activity. J Clin Endocrinol Metab 84:1658–1663 [DOI] [PubMed] [Google Scholar]
  6. Nørrelund H, Nielsen S, Christiansen JS, Jørgensen JO, Møller N 2004 Modulation of basal glucose metabolism and insulin sensitivity by growth hormone and free fatty acids during short-term fasting. Eur J Endocrinol 150:779–787 [DOI] [PubMed] [Google Scholar]
  7. Groop L, Segerlantz M, Bramnert M 2005 Insulin sensitivity in adults with growth hormone deficiency and effect of growth hormone treatment. Horm Res 64(Suppl 3):45–50 [DOI] [PubMed] [Google Scholar]
  8. Krag MB, Gormsen LC, Guo Z, Christiansen JS, Jensen MD, Nielsen S, Jørgensen JO 2007 Growth hormone-induced insulin resistance is associated with increased intramyocellular triglyceride content but unaltered VLDL-triglyceride kinetics. Am J Physiol Endocrinol Metab 292:E920–E927 [DOI] [PubMed] [Google Scholar]
  9. Møller N, Schmitz O, Jøorgensen JO, Astrup J, Bak JF, Christensen SE, Alberti KG, Weeke J 1992 Basal- and insulin-stimulated substrate metabolism in patients with active acromegaly before and after adenomectomy. J Clin Endocrinol Metab 74:1012–1019 [DOI] [PubMed] [Google Scholar]
  10. Nørrelund H, Møller N, Nair KS, Christiansen JS, Jørgensen JO 2001 Continuation of growth hormone (GH) substitution during fasting in GH-deficient patients decreases urea excretion and conserves protein synthesis. J Clin Endocrinol Metab 86:3120–3129 [DOI] [PubMed] [Google Scholar]
  11. Nørrelund H, Nair KS, Jørgensen JO, Christiansen JS, Møller N 2001 The protein-retaining effects of growth hormone during fasting involve inhibition of muscle-protein breakdown. Diabetes 50:96–104 [DOI] [PubMed] [Google Scholar]
  12. Nørrelund H, Nair KS, Nielsen S, Frystyk J, Ivarsen P, Jørgensen JO, Christiansen JS, Møller N 2003 The decisive role of free fatty acids for protein conservation during fasting in humans with and without growth hormone. J Clin Endocrinol Metab 88:4371–4378 [DOI] [PubMed] [Google Scholar]
  13. Møller N, Bagger JP, Schmitz O, Jørgensen JO, Ovesen P, Møller J, Alberti KG, Orskov H 1995 Somatostatin enhances insulin-stimulated glucose uptake in the perfused human forearm. J Clin Endocrinol Metab 80:1789–1793 [DOI] [PubMed] [Google Scholar]
  14. Murray RD, Kim K, Ren SG, Chelly M, Umehara Y, Melmed S 2004 Central and peripheral actions of somatostatin on the growth hormone-IGF-I axis. J Clin Invest 114:349–356 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Leung KC, Doyle N, Ballesteros M, Waters MJ, Ho KK 2000 Insulin regulation of human hepatic growth hormone receptors: divergent effects on biosynthesis and surface translocation. J Clin Endocrinol Metab 85:4712–4720 [DOI] [PubMed] [Google Scholar]
  16. Kopchick JJ, Parkinson C, Stevens EC, Trainer PJ 2002 Growth hormone receptor antagonists: discovery, development, and use in patients with acromegaly. Endocr Rev 23:623–646 [DOI] [PubMed] [Google Scholar]
  17. Trainer PJ, Drake WM, Katznelson L, Freda PU, Herman-Bonert V, van der Lely AJ, Dimaraki EV, Stewart PM, Friend KE, Vance ML, Besser GM, Scarlett JA, Thorner MO, Parkinson C, Klibanski A, Powell JS, Barkan AL, Sheppard MC, Malsonado M, Rose DR, Clemmons DR, Johannsson G, Bengtsson BA, Stavrou S, Kleinberg DL, Cook DM, Phillips LS, Bidlingmaier M, Strasburger CJ, Hackett S, Zib K, Bennett WF, Davis RJ 2000 Treatment of acromegaly with the growth hormone-receptor antagonist pegvisomant. N Engl J Med 342:1171–1177 [DOI] [PubMed] [Google Scholar]
  18. Liu J, Prudom CE, Nass R, Pezzoli SS, Oliveri MC, Johnson ML, Veldhuis P, Gordon DA, Howard AD, Witcher DR, Geysen HM, Gaylinn BD, Thorner MO 2008 Novel ghrelin assays provide evidence for independent regulation of ghrelin acylation and secretion in healthy young men. J Clin Endocrinol Metab 93:1980–1987 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gauna C, Meyler FM, Janssen JA, Delhanty PJ, Abribat T, van Koetsveld P, Hofland LJ, Broglio F, Ghigo E, van der Lely AJ 2004 Administration of acylated ghrelin reduces insulin sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves insulin sensitivity. J Clin Endocrinol Metab 89:5035–5042 [DOI] [PubMed] [Google Scholar]
  20. Broglio F, Gottero C, Prodam F, Gauna C, Muccioli G, Papotti M, Abribat T, Van Der Lely AJ, Ghigo E 2004 Non-acylated ghrelin counteracts the metabolic but not the neuroendocrine response to acylated ghrelin in humans. J Clin Endocrinol Metab 89:3062–3065 [DOI] [PubMed] [Google Scholar]
  21. Thorner MO, Strasburger CJ, Wu Z, Straume M, Bidlingmaier M, Pezzoli SS, Zib K, Scarlett JC, Bennett WF 1999 Growth hormone (GH) receptor blockade with a PEG-modified GH (B2036-PEG) lowers serum insulin-like growth factor-I but does not acutely stimulate serum GH. J Clin Endocrinol Metab 84:2098–2103 [DOI] [PubMed] [Google Scholar]
  22. Jahoor F, Wolfe RR 1987 Reassessment of primed constant-infusion tracer method to measure urea kinetics. Am J Physiol 252:E557–E564 [DOI] [PubMed] [Google Scholar]
  23. Thompson GN, Pacy PJ, Merritt H, Ford GC, Read MA, Cheng KN, Halliday D 1989 Rapid measurement of whole body and forearm protein turnover using a [2H5]phenylalanine model. Am J Physiol 256:E631–E639 [DOI] [PubMed] [Google Scholar]
  24. Nair KS, Ford GC, Ekberg K, Fernqvist-Forbes E, Wahren J 1995 Protein dynamics in whole body and in splanchnic and leg tissues in type I diabetic patients. J Clin Invest 95:2926–2937 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Copeland KC, Nair KS 1994 Acute growth hormone effects on amino acid and lipid metabolism. J Clin Endocrinol Metab 78:1040–1047 [DOI] [PubMed] [Google Scholar]
  26. Wolthers T, Grøfte T, Jørgensen JO, Møller N, Vahl N, Christiansen JS, Vilstrup H 1994 Effects of growth hormone (GH) administration on functional hepatic nitrogen clearance: studies in normal subjects and GH-deficient patients. J Clin Endocrinol Metab 78:1220–1224 [DOI] [PubMed] [Google Scholar]
  27. Ferrannini E 1988 The theoretical bases of indirect calorimetry: a review. Metabolism 37:287–301 [DOI] [PubMed] [Google Scholar]
  28. Jørgensen JO, Jessen N, Pedersen SB, Vestergaard E, Gormsen L, Lund SA, Billestrup N 2006 GH receptor signaling in skeletal muscle and adipose tissue in human subjects following exposure to an intravenous GH bolus. Am J Physiol Endocrinol Metab 291:E899–E905 [DOI] [PubMed] [Google Scholar]
  29. Flyvbjerg A, Bornfeldt KE, Marshall SM, Arnqvist HJ, Orskov H 1990 Kidney IGF-I mRNA in initial renal hypertrophy in experimental diabetes in rats. Diabetologia 33:334–338 [DOI] [PubMed] [Google Scholar]
  30. Skjaerbaek C, Frystyk J, Orskov H, Kissmeyer-Nielsen P, Jensen MB, Laurberg S, Møller N, Flyvbjerg A 1998 Differential changes in free and total insulin-like growth factor I after major, elective abdominal surgery: the possible role of insulin-like growth factor-binding protein-3 proteolysis. J Clin Endocrinol Metab 83:2445–2449 [DOI] [PubMed] [Google Scholar]
  31. Jørgensen JO, Feldt-Rasmussen U, Frystyk J, Chen JW, Kristensen LØ, Hagen C, Ørskov H 2005 Cotreatment of acromegaly with a somatostatin analog and a growth hormone receptor antagonist. J Clin Endocrinol Metab 90:5627–5631 [DOI] [PubMed] [Google Scholar]
  32. Chen JW, Ledet T, Orskov H, Jessen N, Lund S, Whittaker J, De Meyts P, Larsen MB, Christiansen JS, Frystyk J 2003 A highly sensitive and specific assay for determination of IGF-I bioactivity in human serum. Am J Physiol Endocrinol Metab 284:E1149–E1155 [DOI] [PubMed] [Google Scholar]
  33. Møller N, Jørgensen JO 2009 Effects of growth hormone on glucose, lipid, and protein metabolism in human subjects. Endocr Rev 30:152–177 [DOI] [PubMed] [Google Scholar]
  34. Sakharova AA, Horowitz JF, Surya S, Goldenberg N, Harber MP, Symons K, Barkan A 2008 Role of growth hormone in regulating lipolysis, proteolysis and hepatic glucose production during fasting. J Clin Endocrinol Metab 93:2755–2759 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ørskov H, Frystyk J, Nielsen C, Hansen AT, Weeke J, Jørgensen JO 2007 Concomitant, specific determination of growth hormone and pegvisomant in human serum. Growth Horm IGF Res 17:431–434 [DOI] [PubMed] [Google Scholar]
  36. Nørrelund H, Djurhuus C, Jørgensen JO, Nielsen S, Nair KS, Schmitz O, Christiansen JS, Møller N 2003 Effects of GH on urea, glucose and lipid metabolism, and insulin sensitivity during fasting in GH-deficient patients. Am J Physiol Endocrinol Metab 285:E737–E743 [DOI] [PubMed] [Google Scholar]
  37. Miles JM, Haymond MW, Nissen SL, Gerich JE 1983 Effects of free fatty acid availability, glucagon excess, and insulin deficiency on ketone body production in postabsorptive man. J Clin Invest 71:1554–1561 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Keller U, Schnell H, Girard J, Stauffacher W 1984 Effect of physiological elevation of plasma growth hormone levels on ketone body kinetics and lipolysis in normal and acutely insulin-deficient man. Diabetologia 26:103–108 [DOI] [PubMed] [Google Scholar]
  39. Wolthers T, Grøftne T, Møller N, Christiansen JS, Orskov H, Weeke J, Jørgensen JO 1996 Calorigenic effects of growth hormone: the role of thyroid hormones. J Clin Endocrinol Metab 81:1416–1419 [DOI] [PubMed] [Google Scholar]
  40. Møller N, Jørgensen JO, Schmitz O, Møller J, Christiansen J, Alberti KG, Orskov H 1990 Effects of a growth hormone pulse on total and forearm substrate fluxes in humans. Am J Physiol 258:E86–E91 [DOI] [PubMed] [Google Scholar]
  41. Gormsen LC, Jessen N, Gjedsted J, Gjedde S, Nørrelund H, Lund S, Christiansen JS, Nielsen S, Schmitz O, Møller N 2007 Dose-response effects of free fatty acids on glucose and lipid metabolism during somatostatin blockade of growth hormone and insulin in humans. J Clin Endocrinol Metab 92:1834–1842 [DOI] [PubMed] [Google Scholar]
  42. Jørgensen JO, Jessen N, Pedersen SB, Vestergaard E, Gormsen L, Lund SA, Billestrup N 2006 Growth hormone receptor signaling in skeletal muscle and adipose tissue in human subjects following exposure to an intravenous GH bolus. Am J Physiol Endocrinol Metab 291:E899–E905 [DOI] [PubMed] [Google Scholar]
  43. Lucidi P, Parlanti N, Piccioni F, Santeusanio F, De Feo P 2002 Short-term treatment with low doses of recombinant human GH stimulates lipolysis in visceral obese men. J Clin Endocrinol Metab 87:3105–3109 [DOI] [PubMed] [Google Scholar]
  44. Broglio F, Arvat E, Benso A, Gottero C, Muccioli G, Papotti M, van der Lely AJ, Deghenghi R, Ghigo E 2001 Ghrelin, a natural GH secretagogue produced by the stomach, induces hyperglycemia and reduces insulin secretion in humans. J Clin Endocrinol Metab 86:5083–5086 [DOI] [PubMed] [Google Scholar]

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