Growth Hormone (GH) Response to GH-Releasing Peptide-6 in Type ...

0021-972X/98/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1998 by The Endocrine Society

Vol. 83, No. 10 Printed in U.S.A.

Growth Hormone (GH) Response to GH-Releasing Peptide-6 in Type 1 Diabetic Patients with Exaggerated GH-Releasing Hormone-Stimulated GH Secretion* PABLO F. CATALINA, FEDERICO MALLO, M. AMELIA ANDRADE, ´ GUEZ RICARDO V. GARCı´A-MAYOR, AND CARLOS DIE Departments of Endocrinology, Montecelo Hospital, Pontevedra (P.F.C.), and Xeral-Cı´es Hospital, Vigo (M.A.A., R.V.G.-M.). Department of Functional Biology and Health Sciences, University of Vigo (F.M.); and Department of Physiology, University of Santiago de Compostela (C.D.); Spain. ABSTRACT In type 1 diabetes mellitus (DM 1), high GH basal levels and exaggerated GH responses to several stimuli, including GHRH, have been described. GH-releasing peptide-6 (GHRP-6) is a synthetic hexapeptide that specifically stimulates GH release, both in vitro and in vivo. The aim of this study was to evaluate the effects of GHRP-6 alone or in combination with GHRH on GH secretion in DM 1. Six type 1 diabetic males and six age-, sex-, and body mass index-matched control volunteers were studied. Each subject received GHRH (100 mg iv), GHRP-6 (90 mg iv), and GHRH plus GHRP-6 on three separate days. GH peak values were higher in DM 1 patients than in control volunteers, after GHRH (52.2 6 9.8 vs. 19.3 6 6.0 mg/L; P 5 0.016), GHRP-6 (66.2 6 9.6 vs. 39.9 6 6.3 mg/L; P 5 0.05), and GHRH plus

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IGH GH basal levels in patients with type 1 diabetes mellitus (DM 1) were described 3 decades ago (1). For many years, this has been explained as an epiphenomenon of poor metabolic control. In normal subjects, hyperglycemia inhibits pituitary GH response to provocative stimuli. Nevertheless, DM 1 patients, despite elevated blood glucose levels, show exaggerated GH responses to several physiological and pharmacological provocative tests (2–7), and they have mean 24-h GH levels higher than those of normal subjects (8, 9). However, recently it has been shown that, in DM 1, the administration of GH-releasing peptide-6 (GHRP-6) and GHRH (either alone or in combination) induces GH release similar to that in healthy subjects (10). Although GHRH and SRIF have a predominant role in the regulation of the pulsatile pattern of GH secretion, other hypothalamic factors can modulate GH release from the anterior pituitary. GHRP-6 is a synthetic hexapeptide that specifically stimulates GH release, both in vitro and in vivo. This peptide acts in man in a dose-related manner, and it activates pituitary and hypothalamic receptors. It does not operate through GHRH receptors or modulate endogenous GHRH release (11). Received February 5, 1998. Revision received June 18, 1998. Accepted June 30, 1998. Address all correspondence and requests for reprints to: Federico Mallo, Department of Functional Biology and Health Sciences, Faculty of Sciences, Campus of Vigo, University of Vigo, Vigo, Spain E-36.200. * This work was supported by funding from the Fondo de Investigaciones Sanitarias (FIS 96/1739).

GHRP-6 (81.8 6 4.4 vs. 53.7 6 8.2 mg/L; P 5 0.01). An additive GH response to combined administration of these two peptides was observed in diabetic patients. Serum insulin-like growth factor (IGF)-1 levels were diminished in DM 1, with respect to normal subjects (145.2 6 21.5 vs. 269.7 6 42.0 mg/L; P 5 0.01), whereas IGF-binding protein-3 levels were not significantly different between DM-1 and controls. In summary, GHRP-6 is a potent stimulus for GH secretion in DM 1. The combined administration of GHRP-6 plus GHRH constitutes the most powerful stimulus for GH secretion in DM 1. These patients exhibit a greater GH secretory capacity than normal subjects, probably caused by a diminished tone in the IGF-1 sustained negative feedback control exerted upon somatotroph responsiveness. (J Clin Endocrinol Metab 83: 3663–3667, 1998)

The administration of GHRH plus GHRP-6 at maximal doses exerts a synergistic effect on GH secretion in normal subjects. This combined stimulus, which represents the most potent GH releaser to date, is being used to elucidate the physiology of GH neuroregulation, as well as its alterations, in several disease states. Its in vivo synergism suggests that GHRP-6 acts at both pituitary and hypothalamic levels, although the GHRP-6 mechanism of action is unknown. It has been postulated that it may exerts most of its actions at hypothalamic level, perhaps releasing and/or inducing the secretion of an unknown hypothalamic peptide or factor (11–13). The aim of this study was to evaluate the effects of GHRP-6, alone or in combination with GHRH, on GH secretion in a group of DM 1 patients. Subjects and Methods Subjects Six male patients with DM 1, with a mean age of 28.3 yr (range, 24 –35 yr) and a body mass index (BMI) varying from 21.9 –26.2 kg/m2 (mean, 24.3 kg/m2) were studied. The clinical characteristics of the patients are reported in Table 1. All of these patients had normal thyroid and renal function, and none was taking any medication other than insulin. DM 1 subjects followed an appropriate diet and were all taking a mixture of short- and intermediate-acting insulin by sc injections, twice daily. Background retinopathy, determined with retinal fluorescein angiography, was present in two patients. Incipient diabetic nephropathy was detected in the same two patients. None of the patients had other associated diseases. The control group consisted of six normal males with no familiar

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TABLE 1. Clinical characteristics of the subsets studied n

Age (yr)

BMI (kg/m2)

Control

6

DM 1

6

28.6 6 1.1 (26 –32) 28.3 6 2.1 (24 –35)

23.4 6 0.5 (21.2–24.5) 24.3 6 0.7 (21.9 –26.2)

Mean 6

SEM

Duration of disease (months)

180.5 6 42.8 (75–312)

Insulin dose (U/kg z day)

0.71 6 0.05 (0.54 – 0.82)

Hb A1c (%)

4.9 6 0.1 (4.6 –5.3) 9.5 6 0.7 (7.6 –11.8)

(range). Hb A1c, Hemoglobin type A1c.

history of diabetes mellitus. Their mean age was 28.6 yr (range, 26 –35 yr), and their mean BMI was 23.4 kg/m2 (range, 21.2–24.5 kg/m2). All subjects in this group had normal thyroid function and were free of any medication at the time of the study.

Study protocol The experimental protocol was approved by the ethics committee of Xeral-Cı´es Hospital. All subjects were studied after giving informed consent. All tests were performed after habituation to the hospital setting. The experiments started at 0900 h, after an overnight fast, with the subjects recumbent. DM 1 patients did not receive morning insulin injection until the end of each test. Sixty minutes before starting the test, an indwelling catheter was placed in a forearm vein and kept patent by slow 0.9% saline infusion. Each subject underwent three tests, randomly, with an interval of at least 1 week, so each subject served as his own control. After basal samples for measuring GH, insulin-like growth factor (IGF)-1, and IGF-binding protein-3 (IGFBP-3) levels, patients and controls received GHRH, GHRP-6, or GHRH plus GHRP-6 at 0 min, and blood sampling continued until 150 min. GHRH [GRF-(1–29))NH2, Geref Serono, Madrid, Spain] was administered at a dose of 100 mg iv. On a subsequent day, GHRP-6 (Peninsula Laboratories, Inc., Heyerside, UK), formulated and tested at the Complejo Hospitalario de Santiago pharmacy, was administered at a dose of 90 mg iv. On a separate day, GHRH plus GHRP-6, at the above doses, was administered iv.

Assays Serum GH levels were measured in duplicate by a highly sensitive two-site monoclonal antibody immunoradiometric assay (hGH Allegro, Nichols Institute Diagnostics, San Juan Capistrano, CA). The sensitivity of the method is 0.02 mg/L. The mean intraassay coefficients of variation (CVs) were 2.5% (2.6 mg/L), 3.8% (6.9 mg/L), and 2.6% (13 mg/L). The interassay CVs were 2.9% (2.6 mg/L), 3.4% (6.9 mg/L), and 2.5% (13 mg/L). All samples from each subject were analyzed in the same assay. IGF-1 was determined by a commercially available RIA (Nichols Institute Diagnostics). The intra- and interassay CVs were 2.4% (110 mg/L) and 3% (338 mg/L), 3.8% (562 mg/L) and 5.2% (121 mg/L), and 6.5% (371 mg/L) and 6.8% (641 mg/L), respectively. Another commercially available RIA (Mediagnostic GMbH; Tubingen, Germany) was used for the measurement of IGFBP-3 levels. The intraassay CVs were 2.8% (2.2 mg/L) and 3% (4.3 mg/L) and the interassay CVs were 4.8% (2.6 mg/L) and 5.9% (4.5 mg/L). HbA1c levels were measured by high-performance liquid chromatography (Menarini, Italy; normal range , 5.8%).

Statistical analysis Data are presented and analyzed as absolute values, and results are reported as the mean 6 sem. We calculated the peak GH value (maximum GH level measured within 150 min after the stimulus); the mean GH values; the time in which the peak GH value was obtained (T peak), and the AUC (area under the response curve). Friedman’s ANOVA was performed to compare GH levels in each group studied. The MannWhitney test was used for comparisons between different groups. The GH response to each test was also analyzed by the AUC, which was calculated by a trapezoidal method. Undetectable GH levels (,0.02 mg/L) were considered to be equal to 0.05 mg/L for statistical purposes. The statistical level of significance was established at P # 0.05.

Results

The GH responses to the different stimuli are depicted in Fig. 1. The secretory GH parameters obtained in both groups

FIG. 1. Mean 6 SEM. GH responses to GHRH (A), GHRP-6 (B), and GHRH plus GHRP-6 (C), in DM-1 () and control subjects (f). *, P # 0.05; **, P # 0.01.

are shown in Table 2. There were no significant differences in basal GH values between the experiments in both groups. When challenged with a maximal dose of GHRH, normal subjects showed the expected prompt GH peak of 19.3 6 6.0 mg/L. On the other hand, the injection of a maximal dose of GHRP-6 was also a potent GH releaser, with a peak of 39.9 6 6.3 mg/L. The combined administration of both GH secretagogues at the same doses induced a large peak of 53.7 6 8.2 mg/L. In control subjects, the mean GH level after GHRH administration did not differ significantly from that after GHRP-6. The AUC after GHRH administration was 1356.8 6 442.2 mg/Lz150 min and 2349.4 6 441.4 mg/Lz150 min, after GHRP-6 injection [P 5 NS (not significant)]. The administration to these normal volunteers of GHRH plus GHRP-6 at

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TABLE 2. Secretory GH parameters

GHRH

GHRP-6

GHRH 1 GHRP-6

Mean 6

SEM.

Control DM 1 Mann-Whitney (P-value) Control DM 1 Mann-Whitney (P-value) Control DM 1 Mann-Whitney (P-value)

Basal GH (mg/L)

Peak GH (mg/L)

Mean GH (mg/L)

Tpeak (min)

AUC (mg/Lz150 min)

1.5 6 1.0 7.6 6 3.3 NS

19.3 6 6.0 52.2 6 9.8 0.016

10.5 6 3.8 32.9 6 7.6 0.016

35.8 6 6.3 42.5 6 8.4 NS

1356.8 6 442.2 4639.5 6 1018.6 0.006

1.4 6 0.9 5.3 6 3.0 NS

39.9 6 6.3 66.2 6 9.6 0.05

19.3 6 3.5 41.9 6 8.6 0.05

30.8 6 3.8 28.3 6 4.0 NS

2349.4 6 441.4 5480.6 6 1320.0 NS

0.6 6 0.1 7.1 6 4.1 NS

53.7 6 8.2 81.8 6 4.4 0.01

33.2 6 5.0 59.2 6 4.2 0.004

32.5 6 4.2 26.3 6 5.4 NS

4252.2 6 634.0 8141.8 6 844.7 0.007

Comparison by nonparameter Mann Whitney’s test. For description, see Subjects and Methods.

the same doses induced a synergistic GH release (AUC, 4252.2 6 634.0 mg/Lz150 min; P 5 0.04). In DM 1 patients, the GHRH-induced GH peak was 52.2 6 9.8 mg/L, whereas the GHRP-6-induced GH peak was 66.2 6 9.6 mg/L. A peak of 81.8 6 4.4 mg/L was obtained after the combined administration of GHRH plus GHRP-6 (P 5 0.005). When the AUCs were analyzed (mg/Lz150 min), GHRHinduced GH release was 4639.5 6 1018.6, whereas GHRP6-induced secretion was 5480.6 6 1320. The GH discharge after GHRH plus GHRP-6 treatment was 8141.8 6 844.7. There were clear differences in the AUCs when GHRH and GHRP-6 were administered alone, compared with their combined injection (P 5 0.01). The combined administration of both GH releasers showed an additive effect, rather than a synergistic action. When DM 1 patients were compared with the control group, significantly greater GH responses to GHRH, to GHRP-6, and to GHRH plus GHRP-6 were observed in the DM 1 group (Fig. 1). No side effects were reported in any of the subjects tested. Basal serum IGF-1 levels were significantly lower in the DM 1 patients than in the control group (145.2 6 21.5 vs. 269.7 6 42.0 mg/L, respectively; P 5 0.01). We did not find statistical differences in IGFBP-3 values between DM 1 patients and healthy controls (2.7 6 0.9 vs. 3.1 6 0.3 mg/L, respectively; P 5 0.20). Discussion

DM 1 is associated with marked perturbations of the GH/ IGF-1 axis. It has been described as an increased spontaneous GH secretion, but also patients with DM 1 may show exaggerated GH responses to different provocative stimuli such as exercise, arginine, clonidine, dopamine, levodopa, or GHRH (2–7). Twenty-four-hour integrated serum GH concentrations are also elevated in DM 1, because of increases in both the frequency and peak amplitude of GH secretory pulses (8, 9). IGF-1 levels are either normal or reduced, and there is evidence of a reversible defect in IGF-1 generation in these patients (9, 14, 15). This disrupted GH secretion may have relevant physiopathological implications. Several experimental studies suggest the possible relationship between abnormal GH regulation and the development of diabetic microvascular disease (16 –19). Historical studies, both in humans and in animal models, reported the amelioration of

biochemical abnormalities after hypophysectomy or the development of hypopituitarism (20, 21). Moreover, this altered GH secretion may play a potential role in mediating the metabolic derangement of DM 1 (22). Therefore, long-term correction of GH/IGF-1-axis abnormalities in DM 1 may be an important consideration in the development of therapeutic modalities aimed at not only improving metabolic control but also at preventing the pathological sequelae associated with DM 1. Elevated circulating GH levels in subjects with DM 1 have been reported by numerous studies during the past 3 decades (1, 23, 24). The chronic exposure to a hyperglycemic milieu may be important in the pathogenesis of these defects, because circulating glucose level is a potent regulator of GH secretion. Therefore, defects in both glucose and insulin homeostasis may contribute to abnormal regulation of GH in DM 1. At present, the underlying mechanisms responsible for somatotropinergic axis alterations in patients with DM 1 are still not well understood. To gain further insight into mechanisms of altered GH secretion in DM 1, we assessed pituitary GH reserve in these patients, after challenge with the most potent GH-secretagogues available in the clinical setting (namely, GHRH and GHRP-6, administered alone or in combination). Although not a universal finding, some previous studies have found that DM 1 patients exhibited a GH response, after GHRH administration, greater than that of normal subjects (7, 25). These augmented responses to GHRH could not be demonstrated by various other authors (14, 26). Although unclear, at present, these discrepancies could be caused by the known variability in GH responses to GHRH in normal subjects and/or the differences in the patients characteristics, i.e. sex, BMI, adequate metabolic control, etc. Our results confirm that DM 1 patients have an exaggerated GH response to GHRH. In the last few years, GHRP-6 has been employed as a useful tool for exploring GH secretory mechanisms in different states of deranged GH secretion, such as obesity, acromegaly, Cushing syndrome, alterations of growth, or polycystic ovary syndrome, among others (27–31). GHRP-6 activates specific somatotroph and hypothalamic receptors distinct from GHRH receptors (32). It does operate through GHRH-independent mechanisms, and its intracellular signaling system seems to be different from those for GHRH. In

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addition, the combined administration of GHRH plus GHRP-6 represents the most powerful GH secretagogue in most pathophysiological circumstances (13). Confirming previous reports, in our study, a greater GHRP-6-induced GH response was observed in normal subjects (33). When GHRP-6 and GHRH were given together, we observed a synergistic effect, as shown previously (12, 29). This could be explained by the existence of specific receptors for each peptide, at pituitary and hypothalamic levels (32). We also observed that GHRP-6 constitutes a secretagogue of GH secretion, in DM 1 patients, at least as potent as GHRH. Moreover, GH responses to GHRP-6 were higher in DM 1 patients than in normal controls. A GH discharge of such magnitude has never been obtained with other secretagogues in DM 1, despite the multiple stimuli studied (2–7), confirming that GHRP-6 represents one of the most powerful GH secretagogues described to date in DM 1 subjects. It has been reported that acute oral glucose administration blunts the GH response to hexarelin, a GHRP-6 analogue, in normal subjects (34), which might indicate that, indeed, a normal GH response to GHRP-6 in DM 1 patients could also be considered exaggerated. Augmented GHRP-6-induced GH response in DM 1 subjects suggests that GH hypersecretion, in this disease state, is not mediated only by endogenous hypothalamic GHRH or somatostatinergic secretion. Having been proven that GHRP-6 specific binding sites on hypothalamus and pituitary exist (32), it is possible that an endogenous GHRP-like ligand mediates, at least partially, this disarranged GH secretion shown in these patients. Although basal GH secretion is elevated in DM 1 patients, and the ability to respond to both GHRP-6 and GHRH is enhanced, the higher GHRH plus GHRP-6 induced-GH response observed suggests that there is still a considerable reserve of GH in the somatotroph cells ready to be secreted on demand. This fact points out that, in the somatotroph cells of these patients, the GH synthesis might be also augmented. Further in vitro studies will be necessary to confirm this hypothesis. However, we were not able to find the synergistic effect of those peptides when administered together in DM 1, as shown for control volunteers. It seems that this combined administration reaches the maximal capacity of somatotroph responsiveness, both in controls and DM 1 patients (being higher in DM 1). Thus, the additive effect observed in GHRH1GHRP-6-induced GH responses in DM1 is probably caused by the high response elicited by each peptide alone. On the other hand, in a previous report, Vilas-Boas et al. (10) were not able to demonstrate any differences in GH responses to the administration of GHRP-6 and GHRH, either alone or in combination, in DM 1 patients, compared with normal subjects. Moreover, they did not find differences in the responses of GH elicited by GHRH and GHRP-6 in DM1, even in normal volunteers, as would be expected considering previous reports (11, 12). However, they found a synergistic effect of GHRH and GHRP-6 when they where administered together, both in DM 1 and controls. These discrepancies could be explained, because VilasBoas et al. studied a nonsex-matched group (including male and female) and did not find any statistical differences in IGF-1 total serum levels between DM 1 and normal subjects.

Meanwhile, we studied sex-, age- and BMI-matched groups, and the IGF-1 total levels found in DM 1 patients were markedly lower than those found in normal volunteers. Most of our data might be explained by a diminished negative feedback exerted by the circulating IGF-1 levels upon the GH secretion by the pituitary. As shown in Results, we found a reduction in total serum IGF-1 values in DM 1 patients, with respect to controls. This indicates a partial lack of inhibiting control upon somatotroph GH secretion. These data became more relevant when IGF-BP3 levels were shown to be not significantly different in DM 1 from those in control volunteers. Considering that IGF-BP3 binds nearly 90% of circulating IGF-1, the fact that DM 1 showed IGF-BP3 levels similar to controls, and reduced IGF-1 total levels, implies that circulating IGF-1 free-fraction must be markedly reduced in DM 1, in comparison with normal subjects. This could emphasize the fact that a reduction in serum IGF-1 total levels would have marked influence in the physiological control of the somatotrophic axis. In summary, we have shown that DM 1 patients have an elevated GH response to GHRP-6 and GHRH. The combined administration of these two peptides has an additive effect and constitutes the most powerful stimulus for GH secretion in DM 1 subjects. Our data suggest that patients with DM 1 exhibit a greater GH secretory capacity than control subjects, possible caused by a diminished negative feedback control exerted by the IGF-1 upon somatotroph responsiveness. Acknowledgments We are grateful to Aurora Carballo, Marı´a Jose´ Casado, Pilar Cerqueira, and Luis Teniente for their assistance in conducting the experiments and in the analysis of hormonal samples.

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