Low Plasma Ghrelin Level in Gastrectomized Patients Is Accompanied ...

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The Journal of Clinical Endocrinology & Metabolism 90(4):2187–2191 Copyright © 2005 by The Endocrine Society doi: 10.1210/jc.2004-1888

Low Plasma Ghrelin Level in Gastrectomized Patients Is Accompanied by Enhanced Sensitivity to the GhrelinInduced Growth Hormone Release Vera Popovic, Dragana Miljic, Sandra Pekic, Predrag Pesko, Marina Djurovic, Mirjana Doknic, Svetozar Damjanovic, Dragan Micic, Goran Cvijovic, Jovana Glodic, Carlos Dieguez, and Felipe F. Casanueva Institute of Endocrinology, Diabetes, and Metabolism (V.P., D.Mil., S.P., M.Dj., M.Do., S.D., D.Mic., G.C.), Institute of Abdominal Surgery (P.P.), University Clinical Center, Belgrade, Laboratory Consilium (J.G.), 11000 Belgrade, Serbia and Montenegro; and Department of Physiology (C.D.) and Complejo Hospitalario, Endocrine Section (F.F.C.), Faculty of Medicine, Santiago de Compostela University, E-15780 Santiago de Compostela, Spain Ghrelin is a brain-gut peptide with potent GH-releasing activities. It has been suggested that the majority of circulating ghrelin originates from the stomach, with a smaller portion from the small intestine. Gastrectomy (GASTRX) significantly reduces circulating ghrelin concentrations. The implication of decreased circulating ghrelin on the somatotropic axis post GASTRX has not been studied. Therefore, we aimed to investigate the somatotropic axis in 10 gastrectomized patients who underwent total GASTRX for various reasons at least 2 yr ago. At baseline circulating total ghrelin, GH, IGF-I, and IGF binding protein (IGFBP)-3 levels were measured. The GH stimulation test consisted of an insulin-induced hypoglycemia, ghrelin in two iv bolus doses (0.1 and 1 ␮g/kg), and a GHRH test. GH sensitivity was assessed by an IGF-I generation test. All the tests were performed 2 wk apart. At baseline serum ghrelin levels were reduced by 55% in GASTRX patients, compared with the control group (P < 0.05). IGF-I (P < 0.05) and IGFBP-3 (P < 0.01) levels were also significantly lower than in controls. GH response to the insulin-

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HRELIN, THE OREXIGENIC hormone of gastric origin (1, 2) may provide an endocrine link between stomach and central circuits involved with the regulation GH release and energy intake. Ghrelin transfers information from the stomach to the hypothalamus and influences GH release in response to changes in nutritional status (3–5). Circulating ghrelin in weight-stable people correlates negatively with body mass index (BMI) over a wide range and is modulated by alterations in body weight (6, 7). Obesity is characterized by low GH levels as well as low ghrelin levels, but several studies suggest that there is no causative relationship between these two parameters (8). A positive correlation between ghrelin and GH secretion was found in a recent sleep study (9). Nocturnal increase in ghrelin levels shows similarities to GH production, which is promoted by sleep. BeFirst Published Online January 11, 2005 Abbreviations: AUC, Area under the curve; BMI, body mass index; CV, coefficient of variation; GASTRX, gastrectomy; GHS, GH synthetic secretagogue; GHS-R, GHS receptor; IGFBP, IGF binding protein; ITT, insulin tolerance test. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

induced hypoglycemia test in both GASTRX and control subjects was of similar magnitude, whereas circulating plasma ghrelin levels in GASTRX patients were not modified during hypoglycemia. Both doses (0.1 and 1.0 ␮g/kg) of ghrelin stimulated GH release significantly more in GASTRX than control subjects, respectively (peak mean GH ⴞ SE: 18.2 ⴞ 5.6 vs. 5.4 ⴞ 1.3 ␮g/liter, P < 0.03; and 58.7 ⴞ 7.5 vs. 35.3 ⴞ 1.9 ␮g/liter, P < 0.01). There was no difference in GHRH-induced GH response between GASTRX patients and control subjects (P > 0.05). Concomitantly, increased increments in IGF-I and IGFBP-3 to a single bolus of GH were found (P < 0.03). In conclusion, our data suggest that low circulating ghrelin levels, found in GASTRX patients, are accompanied by enhanced ghrelin sensitivity with respect to GH response. This is associated with increased GH responsiveness. GASTRX is a state of acquired chronic hypoghrelinemia that may require replacement with ghrelin, and it is tempting to speculate that this may affect the GH-IGF-IGFBP axis. (J Clin Endocrinol Metab 90: 2187–2191, 2005)

cause ghrelin is an endogenous hormone and its administration is a powerful stimulus to GH release, it is obvious that it might play a role in physiological GH regulation (10, 11). The GH-releasing effect was the first recognized effect of ghrelin, and the GH synthetic secretagogues (GHSs) were actually developed for this particular effect (12). GHSs and ghrelin release GH via a dual mechanism involving the pituitary (13, 14) and hypothalamus (16) by activating a specific receptor (GHS-R) (17–19). The effect of GHSs in vivo is much greater than in vitro. Coadministration of GHS with GHRH causes synergistic GH release (20 –22), and this effect can be used for diagnostic purposes to diagnose adult GH deficiency (23). Centrally and peripherally administered ghrelin stimulates the hypothalamic GHS-R as well as the pituitary GHS-R to release GH. GHSs and ghrelin do not act by altering hypothalamic somatostatin release but rather through functional antagonism with somatostatin (24, 25). GH response to ghrelin in vivo requires an intact endogenous GHRH system, and pituitary stalk lesions cause attenuation of GHS or ghrelin effects (26, 27). There are two postsurgical conditions with decreased circulating ghrelin levels in humans: the Roux-en-Y gastric bypass (28) and gastrectomy (GASTRX). Data on the effects

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of Roux-en-Y gastric bypass on ghrelin levels are disparate (29 –31). Although the stomach is the major source of circulating ghrelin, GASTRX reduces plasma ghrelin concentrations by 65% (32, 33). These findings indicate the compensatory increase in ghrelin secretion from other sites, most probably the intestines. Decreased ghrelin, IGF-I, and IGF binding protein (IGFBP)-3 levels are present in patients with the short bowel syndrome (34). Although the GH-releasing effect was the first recognized effect of ghrelin, physiological studies could not pinpoint the exact role of ghrelin in GH regulation, and the GHS-R knockout models suggest so far only moderate effect (35). Thus we aimed to further explore whether low circulating ghrelin levels would affect the GH-IGF-IGFBP axis in patients with total GASTRX. Subjects and Methods Subjects Ten patients (six males and four females) aged 53.2 ⫾ 5.4 yr (range 44 – 63 yr) with BMI of 22.8 ⫾ 2.5 kg/m2 (range 18.4 –27.9) were included in the study. They all underwent total GASTRX between 1987 and 2002. In six patients the reason for total gastrectomy was adenocarcinoma of the stomach. In the rest the reasons were various: Peutz-Jeghers syndrome, Zollinger-Ellison syndrome, and gastric lymphoma. Ten healthy (six males and four females) age- and BMI-matched subjects were studied as well. The study was approved by the hospital ethical committee. Informed consent was provided from the patients and healthy controls before blood sampling.

Hormonal investigations At baseline after an overnight fast, at 0800 h, samples for ghrelin, GH, IGF-I, and IGFBP-3 were taken, and all samples were stored at ⫺80 C until assayed.

Insulin tolerance test (ITT) After an overnight fast, an iv cannula was inserted at 0800 h, and patients and control subjects underwent a standard ITT (0.15 IU/kg insulin Actrapid, blood glucose ⬍ 2.2 mmol/liter). Samples for GH and ghrelin were taken at 0, 30, 60, 90, and 120 min after a bolus iv insulin injection.

GH effects of ghrelin Patients and control subjects underwent ghrelin administration (1 ␮g/kg bolus iv), and GH was measured in the single 30-min sample (36). Eight GASTRX patients and eight controls further underwent another low-dose ghrelin test (0.1 ␮g/kg bolus iv) 2 wk after the first test.

GHRH test A GHRH test (1 ␮g/kg body weight iv GEREF; Serono, Madrid, Spain) with samples for GH taken at 0, 30, 60, 90, and 120 min was performed in six patients and six control subjects 2 wk apart from the previous test.

IGF-I generation test An IGF-I generation test using a single sc injection of GH 21 IU (7 mg) was performed. Serum IGF-I levels and IGFBP-3 were measured at baseline and 24 h after GH administration to six patients and six healthy subjects. All the tests were performed 2 wk apart, and samples were stored at ⫺80 C until assayed.

Popovic et al. • Hypoghrelinemia and Somatotropic Axis in Gastrectomy

Hormone assays GH was assayed with an immunofluorimetric assay (Wallac, Turku, Finland) with a GH sensitivity of 0.01 ␮g/liter and interassay coefficient of variation (CV) of 6.3% and intrassay CV of 4.2%. Ghrelin (total) was measured with RIA kits (Linco Research, St. Charles, MO) with the limit of detection being 100 pg/ml. Within-assay CV was between 5.6 – 8%. Interassay variations were 7.8 –9.8%. IGF-I levels were measured using chemiluminescent enzyme immunoassay with the Immulite Analyser (Diagnostic Products Corp., Los Angeles, CA) with the limit of detection being 20.0 ng/ml. Within-assay CV was between 2.3 and 3.9%. Interassay variations were 3.7– 8.1%. IGFBP-3 levels were measured using a chemiluminescent enzyme immunoassay with the Immulite analyzer (Diagnostic Products Corp.) with the limit of detection being 20.0 ng/ml. Within-assay CV was between 4.6 and 6%. Interassay variations were 6.8 –9.5%.

Statistical analysis Descriptive statistical data are shown as mean ⫾ sd. Statistical analysis was done using parametric one-way ANOVA and nonparametric Mann-Whitney U and Wilcoxon tests. Analyses were performed using SPSS software (SPSS for Windows, version 10.0, SPSS Inc., Chicago, IL). P ⬍ 0.05 was regarded as indicating statistical significance. Area under the curve (AUC) was calculated using the trapezoidal method.

Results Hormones at baseline

Comparative clinical data in 10 gastrectomized patients and 10 control subjects are summarized in Table l. At baseline, serum ghrelin, IGF-I, and IGFBP-3 levels were significantly lower in GASTRX patients vs. control subjects (Table 1). Mean fasting serum GH concentrations did not differ between GASTRX patients and healthy subjects (0.55 ⫾ 0.85 vs. 0.98 ⫾ 1.89 ␮g/liter; P ⬎ 0.05) ITT

The ITT induced an adequate decrease in circulating glucose levels (nadir 2.2 mmol/liter). After hypoglycemia all patients responded with a peak mean GH 24.9 ⫾ 3.6 ␮g/liter, not different from controls GH 17.8 ⫾ 2.6 ␮g/liter (P ⬎ 0.05; Fig. 1A). The AUCGH in GASTRX patients was 1455 ⫾ 190 ␮g/liter per 120 min and did not differ from the AUCGH 1060 ⫾ 146 ␮g/liter per 120 min (P ⬎ 0.05) in controls. Plasma ghrelin levels did not change significantly during insulin-induced hypoglycemia in GASTRX patients (P ⬎ 0.05; Fig.1B). GH effects of ghrelin

Ghrelin administration in a standard dose (1 ␮g/kg iv) induced prompt and potent GH release in the single 30-min TABLE 1. Comparative clinical and baseline hormonal data in 10 gastrectomized patients and 10 healthy subjects

Age (yr) BMI (kg/m2) Ghrelin (pg/ml) IGF-I (ng/ml) IGFBP-3 (ng/ml) IGF-I/IGFBP-3 molar ratio GH (␮g/liter) a

P ⬍ 0.05;

b

P ⬍ 0.01.

Gastrectomized patients (n ⫽ 10)

Control subjects (n ⫽ 10)

53.2 ⫾ 5.4 22.8 ⫾ 2.5 1204.1 ⫾ 178.4 114.2 ⫾ 20.3 2683 ⫾ 146 0.229 0.55 ⫾ 0.85

52.2 ⫾ 7.4 23.4 ⫾ 4.4 2781.1 ⫾ 968.3a 151.8 ⫾ 24.1a 3910 ⫾ 222b 0.208 0.98 ⫾ 1.89



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potent GH release in GASTRX patients, compared with control subjects (peak means GH ⫾ se: 18.2 ⫾ 5.6 vs. 5.4 ⫾ 1.3 ␮g/liter; P ⬍ 0.03; Fig. 2B). GHRH test

The GH response to the standard GHRH test was not different in GASTRX patients from healthy subjects (peak means GH ⫾ se: 8.32 ⫾ 3.53 vs. 9.07 ⫾ 2.29 ␮g/liter, P ⬎ 0.05; Fig. 3, A and B, respectively). Mean AUCGH after GHRH in GASTRX patients did not differ from control subjects (382.5 ⫾ 147.4 vs. 503.3 ⫾ 136.6 ␮g/liter per 120min, P ⬎ 0.05). IGF-I generation test

FIG. 1. A, GH (micrograms per liter) (mean ⫾ SE) response to ITT in GASTRX patients and healthy controls. B, Ghrelin response in GASTRX patients.

sample, greater than in healthy subjects (peak mean GH ⫾ se: 58.7 ⫾ 7.5 vs. 35.3 ⫾ 1.9 ␮g/liter, P ⬍ 0.01; Fig. 2A). Ghrelin administered in low dose (0.1 ␮g/kg iv) produced a more

FIG. 2. Individual peak GH (micrograms per liter) responses to 1 ␮g/kg ghrelin (A) and 0.1 ␮g/kg ghrelin (B) in GASTRX patients and control subjects.

After a single bolus GH injection (7 mg sc), there was a significant rise in IGF-I in both GASTRX patients and controls (114.2 ⫾ 20.3 to 215.0 ⫾ 21.6 ng/ml vs. 151.8 ⫾ 24.1 to 342.8 ⫾ 35.7 ng/ml) and a significantly larger increment in IGFBP-3 (2683 ⫾ 146 to 3322 ⫾ 141 ng/ml vs. 3910 ⫾ 222 to 4163 ⫾ 279 ng/ml, P ⬍ 0.01) in GASTRX patients vs. controls. The percent increment in IGF-I and IGFBP-3 is shown in Fig. 4, A and B. Serum concentrations of IGFBP-3 increased up to 24% in the GASTRX group, significantly more than in controls (P ⬍ 0.01). Discussion

In this study we have demonstrated that patients with long-term gastrectomy have chronically decreased circulating ghrelin levels. Totally, GASTRX patients have reduced circulating ghrelin concentrations by 55% in our study, compared with the control group. Furthermore, our results demonstrate that gastrectomy induces ghrelin hypersensitivity in terms of GH response. This increased sensitivity is selective to ghrelin because the GH response to GHRH in GASTRX patients was not different

FIG. 3. GH (micrograms per liter) (mean ⫾ SE) response to GHRH test in GASTRX patients (A) and control subjects (B).

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FIG. 4. IGF generation test: the percent changes (mean ⫾ SE) from baseline in IGF-I (A) and IGFBP-3 (B) in GASTRX patients and control subjects. **, P ⬍ 0.01.

from control subjects, which was very recently shown by others (37). The GH effects of the ITT were similar in GASTRX patients and control subjects, and ghrelin levels in GASTRX patients during ITT did not change. Previous findings show that in healthy subjects ghrelin levels during the ITT are suppressed (37, 38). The GH response to insulin-induced hypoglycemia may be part of an integrative stress response rather than a mechanism controlling nutritional status (39). This may be different from the normal fasting situation, when insulin levels are low, perhaps allowing higher tonic circulating ghrelin and thus greater GH secretion. The complexity of the interactions between the classic somatotropic axis and ghrelin (40) prompted us to further explore the putative role of hypoghrelinemia in the GH-IGFIGFBP axis. We found low-normal levels of IGF-I and low IGFBP-3 levels in GASTRX patients. Recently IGF-I levels in the patients with vagotomy were found to be lower than those in normal subjects, which is in accord with our findings (37). It has been suggested that the ratio of IGF-I to its principal binding protein IGFBP-3 provides a measure of biologically active IGF-I (41). The normal IGF-I to IGFBP-3 molar ratio, with low-normal IGF-I and low IGFBP-3 levels, suggests less bioavailable IGF-I. To investigate whether lownormal IGF-I and low IGFBP-3 is due to a change in responsiveness to GH or functional hyposomatotropism, an IGF-I generation test was performed. IGF-I generation tests were developed more than 20 yr ago and are currently used in differentiating GH insensitivity from other disorders characterized by low serum IGF-I, mostly GH deficiency (42). The


increment in IGF-I, and particularly IGFBP-3 after GH administration in GASTRX patients, was significantly greater than in control subjects. Hence, low circulating ghrelin levels parallel increased responsiveness to GH, which may be due to decreased GH production. Increased GH responsiveness to a single bolus dose of GH has also been shown in obese subjects, in whom GH levels are low (43). Whereas it has been reported that both plasma ghrelin and GH levels are low in obesity, no causative relationship has been found in a study in which weight loss increased GH levels and decreased leptin levels, but these changes were not paralleled by increased ghrelin levels (8). On the other hand, along the line that the positive correlations may confirm ghrelin/GH interactions are the recent sleep study (9) and the findings of moderately reduced IGF-I levels in GHS-R null mice (35) and increase in IGF-I after administration of ghrelin mimetics in animals and humans (15, 44, 45). In conclusion, our data suggest that GASTRX is a state of chronic hypoghrelinemia with hypersensitivity to ghrelin in terms of GH response. This seems to be associated with a state of functional hyposomatotropism evidenced by low IGF-I and IGFBP-3 and increased GH responsiveness. Although the underlying mechanisms of these findings remain to be confirmed, it is tempting to speculate that replacement with ghrelin in GASTRX patients will normalize the sensitivity of the GH-IGF-IGFBP axis. Acknowledgments We thank Professor Ghatei Mohammad (Hammersmith Hospital, London, UK) for providing ghrelin. Received September 24, 2004. Accepted January 4, 2005. Address all correspondence and requests for reprints to: Professor Dr. Vera Popovic, M.D., Ph.D., F.R.C.P., Neuroendocrine Unit, Institute of Endocrinology, Diabetes Mellitus and Metabolism, University Clinical Center, Dr Subotic 13, 11000 Belgrade, Serbia. E-mail: [email protected]. This work was supported by Fonda de Investigation Sanitaria, Spanish Ministry of Health, Consoler de Education Junta de Galicia, and the Ministerial Espanola de Cynical y Technologic and Ministry of Science and Technology, Serbia (M1717).

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